Nucleic acid detection technologies and marker molecules in bacterial diagnostics.

Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Chinese University of Hong Kong on 12/26/14 For personal use only.

Review

Nucleic acid detection technologies and marker molecules in bacterial diagnostics Expert Rev. Mol. Diagn. 14(4), 489–500 (2014)

Ott Scheler1, Barry Glynn2 and Ants Kurg*1 1 Department of Biotechnology, IMCB, University of Tartu, Riia 23, Tartu 51010, Estonia 2 Molecular Glycobiotechnology Group, Biochemistry, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland *Author for correspondence: Tel.: +372 737 5018 [email protected]

There is a growing need for quick and reliable methods for microorganism detection and identification worldwide. Although traditional culture-based technologies are trustworthy and accurate at a relatively low cost, they are also time- and labor-consuming and are limited to culturable bacteria. Those weaknesses have created a necessity for alternative technologies that are capable for faster and more precise bacterial identification from medical, food or environmental samples. The most common current approach is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to the specific nucleic acid profiles that are present. This review aims to give an up-to-date overview of different nucleic acid target sequences and respective analytical technologies. KEYWORDS: bacterial target molecules • biosensors • microarrays • microbial diagnostics • next-generation sequencing • nucleic acid diagnostics

Nucleic acid-based bacterial diagnostics

Precise identification of pathogenic agents is needed in clinical diagnostics and in food safety monitoring to treat and/or minimize the effect caused by potentially harmful bacteria. The current ‘gold standard’ for detection and identification of pathogenic bacteria is using culture-based methods. While being reliable and accurate for certain bacteria, these methods may require days and even weeks to get results. In addition, there are several important pathogens that are difficult or impossible to cultivate and can therefore remain undetected by culture-based methods. Nowadays, molecular diagnostics are revolutionizing the clinical management of infectious disease in a wide range of areas, including pathogen detection, evaluation of emerging novel infections, surveillance, early detection of bio-threat agents and antimicrobial resistance profiling as demonstrated by the following overviews [1–6]. This current review concentrates on nucleic acid technologies and marker molecules in bacterial diagnostics. Further reviews describing immunoassay applications to investigate and analyze bacteria are available elsewhere [7,8].

informahealthcare.com

10.1586/14737159.2014.908710

A common approach in nucleic acid diagnostics (NAD) is to examine the nucleic acid sequences of different bacteria for characteristic target regions of each species that can be exploited for their detection and identification in various samples. Either DNA or RNA can be used for bacterial diagnostics, depending on the experiment setup and technological requirements used. Readers are referred to the following reviews for more detailed information [2,9–11]. While DNA is a very stable molecule that can be easily isolated from different biological samples, RNA is labile and easily degraded, especially in nonviable microorganisms. Although more demanding from the perspective of handling, the presence of RNA may sometimes give an insight into the viability of the bacteria under investigation [10,12,13]. The advantages of nucleic acid-based detection over earlier methods include faster turnaround time, reduced handling procedures, and often, improved specificity and sensitivity. Improvements in technology, particularly in the area of in vitro amplification, are also lowering the cost of implementing these methods in a diagnostics laboratory.

Ó 2014 Informa UK Ltd

ISSN 1473-7159

489

Review

Scheler, Glynn & Kurg

Table 1. Summary of selected in vitro amplification technologies in microbial diagnostics.


PCR

Key features

Associated company

Commercial examples

Selected applications

Ref.

PCR, reverse transcriptase PCR (RT-PCR) for RNA amplification

Roche Applied Science

LightCyclerÒ

SeptiFastÒ: real-time PCR for pathogens causing blood stream infections

[111]

Cepheid

GeneXpertÒ

XpertÒ series for molecular diagnostics

[112]

bDNA

Branched-DNA. Isothermal DNA and RNA amplification

Siemens Healthcare

VERSANTÒ 440

Currently available for viral diagnostics and research use only

[113]

HDA

Helicase-dependent amplification: isothermal amplification of DNA

Biohelix

IsoAmpÒ

IsoAmpÒ tHDA kit for custom isothermal DNA amplification

[114]

LAMP

Loop-mediated isothermal amplification: DNA amplification technology

Eiken

LoopampÒ

LoopampÒ detection kits for various environmental and food contaminating bacteria

[115]

LaisionÒ Iam series: currently only different viral infection kits available

[116]

Diasorin

Ò

Laision

LCR

Ligase chain reaction: isothermal DNA amplification technology

None currently available



NASBA

Nucleic acid sequence-based amplification: isothermal RNA amplification

BioMerieux

NucliSENS EasyQÒ

NucliSENS Easy QÒ: system that combines NASBA technology with real-time detection

[117]

RPA

Recombinase polymerase amplification: isothermal amplification of RNA or DNA targets

TwistDx

TwistaÒ

TwistFlowÒ, TwistGlowÒ and TwistAmpÒ kits

[118]

SDA

Strand displacement amplification: isothermal DNA target amplification

Becton Dickinson

BD Probe TecTM

BD ProbeTec ETTM system

[119]

SMART

Signal-mediated amplification of RNA technology: isothermal RNA amplification. Also compatible with DNA amplification




TMA

Transcription mediated amplification: RNA or DNA targets

Hologic GenProbe

TIGRISÒ

APTIMA COMBO 2Ò for detection of urogenital diseases

Detection technologies

The application of nucleic acid sequences for diagnostic purposes has followed the technological advances in molecular biology. These advances include isolation of nucleic acid restriction and amplification enzymes and development of different hybridization techniques. The following section briefly describes established nucleic acid detection technologies and is followed by more in-depth analysis of microarray and biosensor technologies, which are gaining ground in the field of bacterial diagnostics. Established nucleic acid detection technologies

One widely used technology for direct analysis of bacterial nucleic acid sequences is FISH, where labeled DNA probes 490

[120]

hybridize to complementary target sequences in situ [14,15]. FISH has been applied for identification, visualization and localization of various bacteria in many fields of microbiology [16], including clinical microbiology [17,18] and food safety analysis [19]. For a current overview of FISH technologies, readers are directed to the following reviews [20,21]. Even more important from the bacterial diagnostics perspective are nucleic acid in vitro amplification technologies. TABLE 1 includes the list of main amplification technologies and their key features. The most recognized of those technologies is PCR, which was developed in the mid-1980s [22] and has been used in many different formats in microbial diagnostics [23,24] with applications ranging from simple and quick detection of a Expert Rev. Mol. Diagn. 14(4), (2014)


Nucleic acid detection in bacterial diagnostics

single bacterium [25] to more complex multiplex real-time PCR analysis targeting several pathogens [26]. For amplification of RNA targets, reverse-transcriptase PCR has been used [27,28]. There are numerous commercially available PCR-based microbial diagnostics platforms and kits by many different providers. TABLE 1 includes some of the major players. For a more detailed overview of the history, current status and commercial availabilities of PCR-based applications, the following literature is recommended [6,11,23,29–31]. TABLE 1 also lists isothermal enzymatic methods for nucleic acid amplification that do not require active thermocycling. The isothermal nature of those methods offers advantages over PCR with regard to the cost and simplicity in instrumentation and experiment setup. For example, in nucleic acid sequence based amplification (NASBA), cooperation of three different enzymes amplifies target RNA molecules [32]. High RNA specificity makes NASBA less prone to genomic DNA contamination and therefore more suitable for applications where microbial viability can be assessed by target RNA detection, giving it another important advantage over PCR-based methods [13,33]. In microbial diagnostics, detection sensitivities around 1 CFU have been described using NASBA in real-time [34,35] and microarray format [36]. A potential disadvantage of NASBA and similar methods, on the other hand, is that they oblige the user to isolate and detect RNA from the target organism, which is considerably less stable than DNA and more difficult to isolate from patient’s samples. For a thorough review on isothermal nucleic acid amplification and related devices, the readers are referred to the following review [37]. Microarrays

The key advantages that microarray technology has over culture- and PCR-based technologies are high capability for miniaturized multiplexing combined with the ability to detect and identify more than one target molecule simultaneously from the same sample [3,38]. Microbiology and infectious disease diagnostics have consistently contributed 8–9 % of all microarrayrelated publications [39]. Nucleic acids can be analyzed in microarray experiments directly or with an amplification step prior to the hybridization. Several methods have been developed for direct detection of 16S rRNA that is already at the average level of about 10,000–20,000 copies per cell. Such direct detection methods are useful in circumstances where possible amplification-based bias during the detection experiments has to be avoided [40]. Helper oligonucleotides (chaperones) that are complementary to target molecules can be used to increase target binding efficiency [41–43]. Although direct detection methods are easy to implement and do not require additional time-consuming steps that add complexity, target nucleic acids generally require amplification in order to gain sufficient sensitivity and, occasionally, specificity [40]. Many of the amplification technologies listed in TABLE 1 have been used together with microarrays for diagnostic purposes, with PCR-based approaches being the favorite choice. informahealthcare.com

Review

Examples include conventional PCR [44], multiplex PCR [45], multiplex PCR with culture enrichment [46] and reversetranscriptase PCR for RNA targets [47]. From the available isothermal amplification technologies, NASBA has been described in combination with microarray detection [36,48]. One interesting possibility is to use three-dimensional suspension bead arrays as an alternative to two-dimensional, planar arrays. For example, LuminexÒ xMAPTM system consists of 100 different polystyrene microspheres where each one has a specific spectral address. Beads act as the solid support for nucleic acid hybridization reactions allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. Additionally, a fluorochrome coupled to a reporter molecule quantifies the nucleic acid interaction that has occurred at the microsphere surface [49]. Such suspension array technology in combination with target PCR amplification has been used for multiplexed typing of important animal pathogens [50] and is also suggested for the detection of several human pathogens related to biological warfare and terrorism [51]. All the methods described above are based on low-tomedium-density microarrays that typically have a few hundred to a thousand probes immobilized on their surface. These microarrays are suitable for routine clinical diagnostics of infectious diseases because they are easily prepared, inexpensive and require relatively basic equipment for printing, hybridization and scanning. New probe designs or assay layouts can be introduced easily and quickly [52]. In contrast, high-density microarrays are manufactured by in situ oligonucleotide synthesis directly onto the microarray chip. Although more expensive, the advantages are the reproducibility of the manufacturing process, and standardization of reagents, instrumentation and data analysis [53]. A multipathogen identification high-density microarray with 53,660 probes was developed for identification of 18 pathogenic prokaryotes, eukaryotes and viruses. Multiplex-PCR was used to amplify specific DNA sequences that contained pathogenicity and virulence genes, and previously uncharacterized regions. Such simultaneous identification of multiple regions allows an accurate identification of pathogens at species level and their respective hazardousness [54]. In a more recent and ambitious experiment, a high-density microarray was developed for all bacteria and viruses for which full genome sequences were available. Microarray probes were designed to cover both known organisms and also species that have not yet been sequenced but have homology to sequenced organisms. While precise strain or subtype identification was not an initial goal of this experiment, the combined information of multiple probes during the data analysis made it possible. Described array enabled detection and characterization of multiple viruses, phages and bacteria at genus and species levels [55]. Unbiased pathogen detection was demonstrated in another method that was based on whole-genome amplification and resequencing microarrays. In that work, a broad spectrum of bacterial and viral species was identified and antibiotic resistance, pathogenicity and virulence profiles additionally predicted. Identification of a variety of bacteria and viruses, both 491


Review


in isolation and in complex mixtures, was demonstrated. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants [56]. Despite improvements in speed and sensitivity, all of these microarray methods are currently slower than in vitro amplification. In some cases, an amplification step needs to be performed during the microarray workflow. Unless this ‘time to result’ issue is resolved, these methods will not be yet suitable for situations where rapid diagnosis is essential. Biosensors

Biosensors are defined as analytical devices incorporating a biological material, a biologically derived material or a bio-mimic intimately associated with or integrated within a physicochemical transducer or transducing microsystem, which may be optical, electrochemical, thermometric, piezoelectric, magnetic or micromechanical. Biosensors usually yield a digital electronic signal that is proportional to the concentration of a specific analyte or group of analytes. While the signal may in principle be continuous, devices can be configured to yield single measurements to meet specific market requirements [57]. The potential advantages of biosensors over previously described analytical technologies are shorter experiment time, lower cost, easier handling and realtime monitoring capability. The three main transduction principles that have been used for nucleic acid-based biosensing technology are mass, electrochemical and optical sensing [4,58]. An example of a mass-sensitive sensor is quartz crystal microbalance (QCM). QCM allows monitoring of hybridization events using an oscillating piezoelectric crystal with the specific nucleic acid probe molecules immobilized on its surface. The increased mass on the sensor surface due to the hybridization reaction results in a proportional decrease of the oscillating frequency that can be monitored in real time [59]. QCM-based detection of PCR-amplified bacterial target molecules has been described [60,61]. Another mass-sensitive approach is to use small cantilever beams that can be used similarly to QCM as piezoelectric sensor where the added mass causes detectable shift in resonance frequency [62] or, alternatively, the mass-induced bending of the cantilever can be monitored optically [63]. Electrochemical biosensors detect changes in current or potential caused by interactions at the sensor–sample matrix interface. These sensors are classified according to the observed parameter: current, potential or impedance/conductance [4]. In nucleic acids-based bacterial diagnostics, amperometric biosensors have been described for detection of PCR-amplified bacterial toxin genes [64], potentiometric sensors for direct detection of pathogenic bacteria by targeting the 16S rRNA molecules [65] and impedance-based biosensing has been applied to antibiotic resistance detection by targeting short DNA oligonucleotides [66] and longer PCR-amplified DNA fragments [67]. In order to achieve higher sensitivity on electrochemical biosensors for bacterial diagnostics, there have also been suggestions for signal amplification. For example, using enzymatic digestion of probe molecules in combination with target recycling [68]. 492

Optical biosensors are the third main type of biosensors with many different technological subclasses [4,5]. Probably the most widely used of these is surface plasmon resonance (SPR), a refractive index-sensitive label-free technology that enables monitoring of nucleic acid hybridization on sensor surface in real time [69,70]. In bacterial diagnostics, conventional SPR and its highly multiplexed variation SPR imaging (SPRi) have been described for detection and quantification of ribosomal DNA [71] and RNA, respectively [72]. One emerging class of sensitive label-free optical sensors is microring resonators. Their working principle is based on the refractive index sensitivity of optical microcavity structures that are supported by adjacent waveguide arrangements. Microring resonators are highly reproducible, scalable and cost-effective alternatives to other traditional optical sensing technologies due to their easy fabrication process via commercially available semiconductor processing methods. Furthermore, microring sensors require optical technology that is already commonly available in telecommunications, reducing the need for expensive hardware development [73]. Current state-of-the-art technology enables simultaneous monitoring and analysis of 128 different positions in a single microring resonator chip assay [74]. Proof-of-principle microring resonator biosensor has been described for label-free, multiplexed detection and quantification of bacterial transfermessenger RNA (tmRNA) target molecules [75]. So far, sensitivity and time to result are the major disadvantages of biosensor technologies compared with in vitro amplification. Nucleic acid marker molecules in bacterial diagnostics

Within any species or genus of bacteria, there exist unique nucleic acid sequences that can be used to detect and specifically identify these bacteria. Identification of these specific target sequences is the starting point in the development of any NAD assay. In addition to the sequence, there are several other key properties that define an ‘ideal’ NAD target. The target gene (or transcript) should be present in the cell at relatively high copy number, that is, a multicopy target, and it should be present in a wide variety of species while at the same time being sufficiently heterologous at the sequence level to allow for species-specific identification. TABLE 2 presents a selection of analytically relevant bacteria and their corresponding nucleic acid target regions. The reader is also strongly encouraged to read the online SUPPLEMENTARY TABLE (supplementary material can be found online at www.informahealthcare.com/suppl/10.1586/ 14737159.2014.908710) that broadens TABLE 2 by giving an extended list of important pathogens, their corresponding nucleic acid target regions and respective references. The choice of targets and corresponding references has been made to give a broad overview of the topic, and it does not reflect the quality, suitability and performance of particular target sequences. Ribosomal RNA/DNA

One of the most commonly used marker molecules in bacterial diagnostics has been 16S rRNA and its genomic sequence [10,76], as TABLE 2 and SUPPLEMENTARY TABLE appropriately demonstrate. In Expert Rev. Mol. Diagn. 14(4), (2014)


Review


Table 2. A selection of important pathogens and their respective nucleic acid target molecules. Bacillus anthracis

16S rDNA, 16S-23S ITS, pag, rpoB, vvrA

Bacillus cereus

16S rDNA, 16S-23S ITS, 23S rDNA, hly, rpoB, vvrA

Campylobacter

16S rDNA/rRNA, 16S-23S ITS, 23S rRNA, ceuE, fla genes, fur, glyA, groESL genes (hsp60), gyrB

Campylobacter jejuni

16S rDNA, 16S-23S ITS, 23S rDNA, cdt genes, fur, glyA, ceu genes, groESL genes (hsp60), gyrA, hipO, mapA, oxido-reductase

Chlamydophila pneumoniae

16S rDNA, 16S-23S ITS, argR, fus genes, gyrA, momp, ompA

Clostridium botulinum

16S rDNA, 23S rDNA, BoNT genes

Escherichia coli

16S rDNA/rRNA, 23S rDNA, clpB, csp, fimH, fus genes, gyrA, gyrB, hlyA, lacZ, rnr, rpoN, ssrA (tmRNA), wzx, wzy

Haemophilus influenzae

16S rDNA/rRNA, 23S rDNA, bexA, cap genes, frdB, fucK, fus genes, gyrA/B, HI, IS1016, ompP6, rnpB,

Helicobacter pylori

16S rDNA, 23S rDNA, cag genes, cpn60, rpoB, ureC, vacA

Klebsiella pneumoniae

16S rDNA/rRNA, 16S-23S ITS, 23S rDNA, fimK, fus genes, gyrA, KPC genes

Legionella pneumophila

16S rDNA/rRNA, dotA, fus genes, gyrA, gyrB, mip, ssrA (tmRNA), wzm

Listeria monocytogenes

16S rDNA, 23S rDNA, clp genes, dth-18, gyrB, hly, iap, inl genes, plc genes, prfA, ssrA (tmRNA)

Moraxella catarrhalis

16S rDNA, 16S-23S ITS, copB, fus genes, gyrA/B, uspA

Mycobacterium tuberculosis

16S rDNA/rRNA, 16S-23S ITS, IS6110, gyrA/B, katG, rpoB

Mycoplasma pneumoniae

16S rDNA, 23S rDNA, CARDS tx, dnaK, fus genes, gyrA, mp3, p1, pdhA, tuf

Neisseria meningitidis

16S rDNA/rRNA, cap, cnl loci, ctrA, galE/lipA, IS1106, lcbB, porA, sac genes, Sg, synG, siaD, wnm, xcbA

Proteus vulgaris

16S-23S ITS, 23S rDNA, fus genes, gyrA

Pseudomonas aeruginosa

16S rDNA/rRNA, 16S-23S ITS, 23S rDNA, algD, ecfX, ETA, fliC, gyrB, opr genes

Salmonella

16S rDNA, 16S-23S ITS, gyrB, hilA, invA, IS200, ogdH, ssrA (tmRNA)

Salmonella enterica

16S rDNA, 16S-23S ITS, 23S rDNA/rRNA, gyrB, hilA, iagA, invA, sdfI

Shigella

16S rRNA, 23S rDNA, gyrB, ipaH, IS1, lacY, uidA, virA, wfaU, wzx, wzy

Staphylococcus aureus

16S rDNA/rRNA, 16S-23S ITS, 23S rDNA, fus genes, gyrA/B, mecA, nuc, orfX-SCCmec, SE genes, ssrA (tmRNA), tuf

Streptococcus agalactiae (Group B)

16S rDNA/rRNA, cfb, cps genes, ptsI, scpB, ssrA (tmRNA)

Streptococcus pneumoniae

16S rDNA/rRNA, 16S-23S ITS, 23S rDNA, cps genes, fus, gyrA, 16S-23S ITS, gyrB, lytA, parE, ply, psaA, recA, rpoB, Spn9802, Spn9828, ssrA (tmRNA), tuf

Vibrio cholerae

16S rDNA, 16S-23S ITS, 23S rDNA, ctx genes, dnaJ, groEL, lolB, ompU, rfbE, rtx, sodB, tcp genes, toxR

Yersinia pestis

16S rDNA, caf1, entF3, pCD1, IS genes, lcrV, pla, yihN, ymt

A full list of bacteria and their target regions with the corresponding analysis technologies and references can be found in the ITS: Internal transcribed spacer; tmRNA: Transfer-messenger RNA.

addition to meeting the defined key properties, 16S rRNA sequences have already been described for almost all of the known bacteria allowing for a quick and relatively easy probe design for various detection platforms [20]. For example, first microarray publication for bacterial diagnostics described detection and differentiation between bacteria by targeting the respective 16S rRNA molecule [77]. In a recent work, next-generation sequencing (NGS) was utilized for molecular diagnosis of a patient with severe bacterial informahealthcare.com

SUPPLEMENTARY TABLE.

infection. Patient biopsy sample was analyzed for the presence of pathogenic bacteria by deep sequencing the 16S rRNA genes present. This work clearly demonstrates the necessity to adapt quickly novel analytical technologies in microbial diagnostics that address the shortcomings of culturing and conventional molecular methods. In this case, NGS enabled correct identification and treatment against the pathogen that caused severe longtime damage to the patient when conventional methods failed. The case illustrates several capabilities of deep sequencing as a 493


Review


clinical diagnostic tool by targeting 16S rRNA. Specifically, discovering the identity of individual organisms within polymicrobial samples, classifying organisms directly from patient specimens without the need for culture and characterizing nonviable or unculturable organisms [78]. However, the ubiquitous nature and sequence conservation of bacterial 16s rRNA represent both an advantage and a disadvantage in species identification. In the first instance, it is possible to design primers that will amplify this gene from a broad range of pathogenic organisms. On the other hand, the high degree of sequence conservation in the rRNA genes can mean it is relatively difficult to distinguish closely related species within certain genus [79]. For example, it has been reported that in case of the genus Streptococcus, the 16S rRNA gene sequence is not variable enough to allow identification of closely related species or subspecies [80]. Similar situations have been described for other bacterial taxa [81–85]. Another difficulty is the presence of multiple copies of nonidentical rRNA genes in certain bacterial genomes. Such intragenomic heterogeneity may further hinder the use of this target for precise diagnostic applications [85–87]. Low differentiation power of this marker molecule within certain genera combined with copy number heterogeneity has driven analytical scientists to look for alternative marker molecules that can be used for more accurate bacterial diagnostics in these cases. One considered alternative to overcome the low discriminating power of highly conserved 16S rRNA/rDNA is to use 23S rRNA or its gene sequence. While possessing the same positive characteristics as described for 16S rRNA, the 23S rRNA molecule has more variation between species, including those of medical and food safety importance [44,88]. Another possible choice are the intergenic spacer regions (ISR) (also known as internal transcribed spacer) between the genes in the ribosomal operon (rrn) [89,90]. The intergenic region between 16S and 23S rRNA (internal transcribed spacer 1) has been used most in bacterial analysis as it contains hyper-variable regions that should theoretically allow differentiation even between strains [47,91]. ISR sequences have disadvantages too. Multiple ribosomal operons are present within most bacterial genomes, and as previously described with 16S rDNA, variation of ISR sequences between these copies has been described [89,90]. Unless such intragenomic heterogeneity of ribosomal genes is considered while developing the diagnostics assay, correct typing of many bacterial species or strains remains complicated [92]. Nonribosomal nucleic acid marker molecules

While ribosomal gene targets have demonstrated considerable utility when used in diagnostics, the disadvantages associated with their use for some organisms have warranted the investigation of alternative targets. The bacterial ssrA gene that encodes tmRNA is another example of a gene with both conserved and variable regions that can be exploited for species identification. It is present in all bacterial species and is expressed at relatively high levels [83,93]. Escherichia coli has previously been shown to have approximately 500 tmRNA 494

molecules per cell [94] while quantitative real-time PCR and in vitro transcribed RNA standards have been used to show that tmRNA is present from 300 to 2000 copies per cell in a variety of bacterial pathogens [95]. The high copy number of tmRNA is advantageous in developing NAD assays while the use of an RNA target has the potential to enable viable pathogens to be distinguished from nonviable organisms. Additionally, tmRNA contains sufficient sequence heterogeneity between species to permit the development of specific DNA probes and pathogen-specific NAD tests [36,83]. These targets are not suitable in situations where the ssrA (or rDNA genes) are conserved between closely related species. For example, many species of the Mycobacterium genus are indistinguishable using either tmRNA or rRNA. In this case, the tuf gene has been successfully used for identification [96]. GyrB was also found to be a very promising and useful alternative target for identification and taxonomic analysis for the Bacillus subtilis group that consists of eight very closely related species [97]. Similar approaches have been used to overcome limitations of rRNA-based diagnostic using a variety of additional targets, including but not limited to gyrB for various Cronobacter species [98], rpoB for Legionella [99] or even a combination of genes for some species including Acetinobacter spp. [100]. In addition to describing bacterial composition of investigated environment or biological sample, it is often also important to know what kind of processes those bacteria can perform and/or how dangerous they are. Analysis of functional gene markers that determine their virulence should give a better understanding of microbial communities and their responses/ adaptations to surrounding environment and susceptibility for treatment. TABLE 2 and complementary SUPPLEMENTARY TABLE give a more thorough overview of the target molecules that are recommended and used for bacterial identification. Multispecies & multilocus targeting in bacterial diagnostics

In order to achieve a wider range of specificity encompassing many different bacterial taxa, several marker molecules can be used simultaneously in a single microarray platform [101]. Additionally, analysis of different bacterial genes responsible for virulence and resistance to antibiotics has been demonstrated on microarray technology [102], combination of multiplex-PCR with microarray-based detection [103] and PCR-biosensor combination [67]. An interesting option is to use nucleic acid probe sequences that do not target certain gene or RNA molecules, but some unique nucleotide patterns that are present in bacterial genome. While designing such probes, the annotation and the biological background of a target region is usually irrelevant. Such analytical microarrays have been described for the detection of all known viruses and bacteria with complete genome sequence available [55]. In another work, a comparative genomic approach was used in developing a microarray with specific probes for 11 major food-borne pathogens [104]. Laboratories worldwide are also enthusiastically developing different NGS technologies for clinical microbiology purposes. High-throughput sequencing was initially confined to specialized Expert Rev. Mol. Diagn. 14(4), (2014)



laboratories due to the need for highly specialized personnel and due to the high costs of instruments and supplies, but the recent arrival of less-expensive platforms and the simplification of the protocols have fostered the rapid introduction of NGS in microbiology laboratories [105]. For example, NGS was implemented to high-throughput multilocus sequence typing of mixed samples of four pathogenic bacteria by sequence analysis of several housekeeping genes. The described experiment included amplification and sequencing of target genes from 575 different isolates of these four pathogens [106]. Whole-genome targeting

Ultimately, the whole genome of the bacterium can act as a marker sequence, providing highest possible level of information about its metabolism, as well as clinically relevant information regarding antibiotic resistance, virulence and toxicity. The primary advantage of this approach is that it allows multiple parameters of a bacterial infection to be investigated simultaneously such as species identification, strain type, pathogenicity and antimicrobial resistance profiles. Time to result is still too slow compared with in vitro amplification meaning that this approach is currently unsuitable for a rapid diagnostics. However, technological improvements may see this approach becoming more acceptable for rapid bacterial identification. NGS technologies already enable increasingly cheaper and quicker production of bacterial genome data, accompanied by vast enlargement of corresponding databases for storage and analysis of the corresponding data [107]. It has been predicted that in near future a routine diagnostics scenario will include direct sequencing of whole genomes of a bacterial population and comparison of obtained results against databases containing well-annotated data giving necessary information for future treatment of patients and other actions [108]. As a herald of future clinical diagnostics and epidemiological studies, high-throughput genome sequencing was applied to investigate the putative multidrug-resistant E. coli outbreak in the neonatal unit of a hospital. Four suspected outbreak strains were sequenced together with another phenotypically similar pathogenic E. coli strain genome as a comparison. Genome-wide single-nucleotide polymorphism analysis demonstrated that the four neonatal intensive care unit strains were identical and also easily differentiated from the comparator strain. NGS data revealed the exact genetic determinants that coded resistance to several antibiotics. In addition, this work also demonstrated the presence of significantly different antimicrobial resistance genes in outbreak and comparison strains that had previously shown similar results in antibiotic sensitivity testing [109]. Another promising option is to design microarrays that contain probes without any prior knowledge about the target bacterium or its genome sequence. A high-density ‘universal bio-signature detection array’ was developed that contained probes complementary to every possible 9-mer oligonucleotide sequence (49 = 262144 probes). Each genome hybridized onto the probes on this array has a unique pattern of signal intensities. These data can be used to affiliate investigated samples into known phylogenomic relationships informahealthcare.com

Review

even with mixed analytical samples. Data gathered from the universal bio-signature detection array microarray experiments could also be used for analyzing phylogenetic relationships between different organisms [110]. Improvements in speed and sensitivity and a simplification of instrument requirements are required before this approach can be used for routine diagnostics. While all these techniques in this section are interesting and provide useful information, they are slow and often relatively insensitive compared with conventional methods. Moreover, microarray and NGS detection technologies require specialized and expensive equipment for both hybridization and detection. In order to become useful in a clinical diagnostics setting, these technologies will need to improve in both speed and sensitivity and at least match the standards currently set by in vitro amplification-based methods. Expert commentary & five-year view

The foundations of any nucleic acid test in microbial diagnostics are the unique target sequences that are detected and identified using a suitable analytical technology. Existing approaches are constantly being combined with emerging technologies and platforms in order to develop better systems for routine use. The definite trend and requirement is to develop systems that are simpler, more cost-effective and easier to use while also being capable of multianalyte detection with little sample preparation and fast turnaround time. Most current NAD include in vitro amplification with PCR being the dominant technology. Emerging and next-generation technologies such as array-based technologies and biosensing devices are being developed to complement and improve those technologies. Micro- and nanoscale fabrication methods are being applied to develop rapid, miniaturized lab-on-a-chip platforms for pathogen detection. The advantages of microscaling include reduced manufacturing and assay costs due to lower assay volumes, which may also have a long-term environmental benefit in lowering the amounts of chemicals required per assay. Use of micro (or nano) fluidics will result in shorter assays due to faster assay mixing rates in the reduced volumes. In addition, different electrochemical and optical detection methods can be incorporated into these miniaturized devices. That is attractive in analytical context as these technologies couple a label-free hybridization signal with instrument readout in real time. These technologies are paving the ground for assay formats that will not require labeled reporter molecules, such as enzymes or fluorophores. However, to gain significant market share, these analytical systems will have to at least match the sensitivity and specificity of in vitro amplification assays. Only when this is achieved will the advantages in speed, sensitivity and cost come to the fore. Specific nucleic acid target regions will remain central to NAD assays. As demonstrated by the marker molecule section of this article in general, there is no ‘ideal’ marker molecule that has been identified yet; therefore, several alternative molecules and approaches are being used for bacterial detection and identification. We predict that ribosomal RNA and its genomic 495


Review


regions will continue with great popularity, but on a smaller scale. Alternative target molecules and regions that give more information of present pathogens will definitely gain ground. Eventually, the analysis of whole-genome data of the bacteria will become commonplace in diagnostics, providing the highest possible level of information about its metabolism as well as clinical relevance regarding antibiotics susceptibility and resistance, virulence and toxicity. In time, future technologies will move NAD away from centralized laboratory facilities and closer to the patient. These future locations will include emergency rooms, physician’s offices and potentially even the home. Such point-of-care instruments may also facilitate in situ analysis of food and drink samples and also decentralized environmental analysis. Further advances can be anticipated in surveillance and detection of bio-threat agents. Centralized laboratories will remain important but with different priorities and tasks. For example, centralized laboratories may become more involved in the application of next-generation

sequencing for such routine diagnostics scenarios where whole genomes of a bacterial population will be sequenced and analyzed according to the information in annotation databases. They will also become a hub of information retrieval, storage and analysis that communicates with point-of-patient (referring to at-home or physician’s offices) testing systems, allowing, for example, remote handling of several patients by clinicians. Financial & competing interests disclosure

O Scheler and A Kurg acknowledge support from SF0180027s10 grant from Estonian Ministry of Education and Research. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

Key issues • Established methods (especially culture-based methods) have over a century of development and tradition behind them. Expecting an emerging technology to fully replace these methods in a short time frame is unrealistic whatever the demonstrated improvements in speed and precision. • Nucleic acid diagnostics for microbial pathogens initially relied on PCR-based technologies. This established the gold standard in terms of sensitivity and specificity against which all subsequent technologies are measured. • Alternative nucleic acid amplification technologies to PCR have made steady increases in their popularity as some of them have demonstrated sensitivity comparable to PCR. They have become popular in the development of point-of-care diagnostics systems due to simplified instrument requirements. • Microarray-based methods are advantageous as they offer the possibility of highly parallel assays being included on a single array. Disadvantages include the requirement to include a pre-hybridization amplification step, a labeling step and typically a complex and expensive imaging system. • Biosensor technologies have the primary advantage of detection of unlabeled nucleic acid targets in a multiplex assay format. Current systems compare poorly to amplification-based technologies in terms of sensitivity, but technology advances will overcome this difficulty. • Combination of a brief isothermal amplification with a robust biosensor detection system may ultimately lead to the next wave of diagnostics platforms particularly for point-of-care and resource-limited scenarios. • Close alignment with biomedical diagnostics and the electronics fabrication industry will see improvements in miniaturization of assay platforms as well as significant reductions in manufacture costs. The microring resonator biosensors described in this review are an initial example of this convergence. • Specific nucleic acid target regions will remain central to nucleic acid diagnostics assays. Ribosomal RNA continues with great popularity, but on a smaller scale as other target regions are used more. • The improvements in massively parallel next-generation sequencing technologies over the next 5–10 years will allow clinicians to routinely profile pathogenic organisms in extreme detail (including species, strain, pathogenicity and resistance profiles).

References

3.

Papers of special note have been highlighted as: • of interest 1.

Jasson V, Jacxsens L, Luning P, et al. Alternative microbial methods: an overview and selection criteria. Food Microbiol 2010; 27(6):710-30

2.

Barken K, Haagensen J, Tolker-Nielsen T. Advances in nucleic acid-based diagnostics of bacterial infections. Clin Chim Acta 2007;384(1-2):1-11

496

4.

Kostic T, Francois P, Bodrossy L, Schrenzel J. Oligonucleotide and DNA microarrays: versatile tools for rapid bacterial diagnostics. In: Zourob M, Elwary S, Turner A, editors. Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems. Springer, New York, NY, US; 2008. p. 629-58 Lazcka O, Del Campo F, Munõz F. Pathogen detection: a perspective of

traditional methods and biosensors. Biosens Bioelectron 2007;22(7):1205-17 •

Comprehensive review of traditional methods in pathogen detection and their comparison to biosensors with a perspective to the future.

5.

Velusamy V, Arshak K, Korostynska O, et al. An overview of foodborne pathogen detection: in the perspective of biosensors. Biotechnol Adv 2010;28(2): 232-54

Expert Rev. Mol. Diagn. 14(4), (2014)


6.


7.

8.

Yang S, Rothman RE. Review PCR-based diagnostics for infectious diseases : uses, limitations, and future applications in acute-care settings. Lancet Infect Dis 2004;4:337-48 Andreotti P, Ludwig G, Peruski A, et al. Immunoassay of infectious agents. Biotechniques 2003;35(4):850-9 Banada PP, Bhunia AK. Antibodies and immunoassays for detection of bacterial pathogens. In: Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems. Springer, New York, NY, US; 2008. p. 567-602

9.

Ludwig W. Nucleic acid techniques in bacterial systematics and identification. Int J Food Microbiol 2007;120(3): 225-36

10.

O’Connor L, Glynn B. Recent advances in the development of nucleic acid diagnostics. Expert Rev Med Devices 2010;7(4):529-39

11.

•

12.

Sibley C, Peirano G, Church D. Molecular methods for pathogen and microbial community detection and characterization: Current and potential application in diagnostic microbiology. Infect Genet Evol 2012;12(3):505-21 Good review of current status in nucleic acid detection technologies for bacterial diagnostics. Birch L, Dawson C, Cornett J, Keer J. A comparison of nucleic acid amplification techniques for the assessment of bacterial viability. Lett Appl Microbiol 2001;33(4): 296-301

13.

Keer J, Birch L. Molecular methods for the assessment of bacterial viability. J Microbiol Methods 2003;53(2):175-83

14.

DeLong E, Wickham G, Pace N. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science 1989;243(4896):1360-3

15.

Amann R, Krumholz L, Stahl D. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 1990;172(2):762-70

16.

Amann R, Fuchs B, Behrens S. The identification of microorganisms by fluorescence in situ hybridisation. Curr Opin Biotechnol 2001;12(3):231-6

17.

Kempf V, Trebesius K, Autenrieth I. Fluorescent In situ hybridization allows rapid identification of microorganisms in blood cultures. J Clin Microbiol 2000; 38(2):830-8

18.

19.

Peters R, Savelkoul P, Simoons-Smit A, et al. Faster Identification of Pathogens in


Positive Blood Cultures by Fluorescence In Situ Hybridization in Routine Practice. J Clin Microbiol 2006;44(1):119-23

30.

Schuller M, Sloots TP, James GS, et al., editors. PCR for clinical microbiology. Springer, Netherlands, NL; 2010

Schmid M, Lehner A, Stephan R, et al. Development and application of oligonucleotide probes for in situ detection of thermotolerant Campylobacter in chicken faecal and liver samples. Int J Food Microbiol 2005;105(2):245-55

31.

Maurer J. Rapid detection and limitations of molecular techniques. Annu Rev Food Sci Technol 2011;2:259-79

32.

Compton J. Nucleic acid sequence-based amplification. Nature 1991;350(6313): 91-2

33.

Mader A, Riehle U, Brandstetter T, et al. Microarray-based amplification and detection of RNA by nucleic acid sequence based amplification. Anal Bioanal Chem 2010;397(8):3533-41

34.

Loens K, Beck T, Goossens H, et al. Development of conventional and real-time NASBA for the detection of Legionella species in respiratory specimens. J Microbiol Methods 2006;67(3):408-15

35.

O’Grady J, Lacey K, Glynn B, et al. tmRNA–a novel high-copy-number RNA diagnostic target–its application for Staphylococcus aureus detection using real-time NASBA. FEMS Microbiol Lett 2009;301(2):218-23

36.

Scheler O, Kaplinski L, Glynn B, et al. Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes. BMC Biotechnol 2011; 11(1):17

37.

Chang C-C, Chen C-C, Wei S-C, et al. Diagnostic devices for isothermal nucleic acid amplification. Sensors 2012;12(6): 8319-37

•

Extensive review of different isothermal nucleic acid amplification technologies and related devices.

38.

Severgnini M, Cremonesi P, Consolandi C, et al. Advances in DNA microarray technology for the detection of foodborne pathogens. Food Bioprocess Technol 2011; 4(6):936-53

39.

Miller M, Tang Y-W. Basic concepts of microarrays and potential applications in clinical microbiology. Clin Microbiol Rev 2009;22(4):611-33

40.

Leski T, Malanoski A, Stenger D, Lin B. Target amplification for broad spectrum microbial diagnostics and detection. Future Microbiol 2010;5(2):191-203

41.

Chandler D, Newton G, Small J, Daly D. Sequence versus structure for the direct detection of 16S rRNA on planar oligonucleotide microarrays. Appl Environ Microbiol 2003;69(5):2950-8

42.

Small J, Call D, Brockman F, et al. Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays.

20.

Amann R, Fuchs B. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol 2008;6(5): 339-48

21.

Wagner M, Haider S. New trends in fluorescence in situ hybridization for identification and functional analyses of microbes. Curr Opin Biotechnol 2012; 23(1):96-102

22.

Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 1985;230(4732):1350-4

23.

Espy M, Uhl J, Sloan L, et al. Real-time PCR in clinical microbiology: applications for routine laboratory testing. Clin Microbiol Rev 2006;19(1):165-256

24.

25.

26.

27.

28.

29.

Review

Postollec F, Falentin H, Pavan S, et al. Recent advances in quantitative PCR (qPCR) applications in food microbiology. Food Microbiol 2011;28(5):848-61 Uhl J, Adamson S, Vetter E, et al. Comparison of lightcycler PCR, rapid antigen immunoassay, and culture for detection of group a streptococci from throat swabs. J Clin Microbiol 2003;41(1): 242-9 Thurman K, Warner A, Cowart K, et al. Detection of Mycoplasma pneumoniae, Chlamydia pneumoniae, and Legionella spp. in clinical specimens using a single-tube multiplex real-time PCR assay. Diagn Microbiol Infect Dis 2011;70(1):1-9 Klein P, Juneja V. Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR. Appl Environ Microbiol 1997;63(11):4441-8 Fey A, Eichler S, Flavier S, et al. Establishment of a real-time PCR-based approach for accurate quantification of bacterial RNA targets in water, using salmonella as a model organism. Appl Environ Microbiol 2004;70(6): 3618-23 Weile J, Knabbe C. Current applications and future trends of molecular diagnostics in clinical bacteriology. Anal Bioanal Chem 2009;394(3):731-42

497

Review


Appl Environ Microbiol 2001;67(10): 4708-16 43.


44.

45.

46.

47.

Kaplinski L, Scheler O, Parkel S, et al. Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides. BMC Biotechnol 2010;10:34 Anthony R, Brown T, French G. Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array. J Clin Microbiol 2000;38(2):781-8 Volokhov D, Rasooly A, Chumakov K, Chizhikov V. Identification of Listeria Species by Microarray-Based Assay. J Clin Microbiol 2002;40(12):4720-8 Panicker G, Call D, Krug M, Bej A. Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays. Appl Environ Microbiol 2004;70(12):7436-44 Weber D, Sahm K, Polen T, et al. Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria. J Appl Microbiol 2008; 105(4):951-62

48.

Scheler O, Glynn B, Parkel S, et al. Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications. BMC Biotechnol 2009;9:45

49.

Dunbar S. Applications of Luminex xMAP technology for rapid, highthroughput multiplexed nucleic acid detection. Clin Chim Acta 2006;363(1-2): 71-82

50.

Gastaldelli M, Stefani E, Lettini A, Pozzato N. Multiplexed typing of Mycobacterium avium subsp. paratuberculosis types I, II, and III by Luminex xMAP suspension array. J Clin Microbiol 2011;49(1):389-91

51.

Schweighardt A, Battaglia A, Wallace M. Detection of Anthrax and Other Pathogens Using a Unique Liquid Array Technology. J Forensic Sci 2013; In press

52.

Mikhailovich V, Gryadunov D, Kolchinsky A, et al. DNA microarrays in the clinic: infectious diseases. Bioessays 2008;30(7):673-82

53.

Dalma-Weiszhausz D, Warrington J, Tanimoto E, Miyada C. The affymetrix GeneChip platform: an overview. Methods Enzymol 2006;410(06):3-28

54.

Wilson W, Strout C, DeSantis T, et al. Sequence-specific identification of 18 pathogenic microorganisms using microarray technology. Mol Cell Probes 2002;16(2):119-27

498

55.

Gardner S, Jaing C, McLoughlin K, Slezak T. A microbial detection array (MDA) for viral and bacterial detection. BMC Genomics 2010;11(1):668

•

This article describes the design and application of high-density pan-microbial microarray that was intended to detect all known viruses, bacteria and plasmids.

56.

Berthet N, Dickinson P, Filliol I, et al. Massively parallel pathogen identification using high-density microarrays. Microb Biotechnol 2008;1(1):79-86

57.

Biosensors World Congress. Available from: www.biosensors-congress.elsevier.com

58.

Arora P, Sindhu A, Dilbaghi N, Chaudhury A. Biosensors as innovative tools for the detection of food borne pathogens. Biosens Bioelectron 2011;28(1):1-12

59.

60.

61.

62.

O’Sullivan C, Guilbault G. Commercial quartz crystal microbalances – theory and applications. Biosens Bioelectron 1999; 14(8-9):663-70 Mo X-T, Zhou Y-P, Lei H, Deng L. Microbalance-DNA probe method for the detection of specific bacteria in water. Enzyme Microb Technol 2002;30:583-9 Wu V, Chen S-H, Lin C-S. Real-time detection of Escherichia coli O157: H7 sequences using a circulating-flow system of quartz crystal microbalance. Biosens Bioelectron 2007;22(12):2967-75 Hwang K, Lee S, Eom K, et al. Nanomechanical microcantilever operated in vibration modes with use of RNA aptamer as receptor molecules for label-free detection of HCV helicase. Biosens Bioelectron 2007; 23(4):459-65

for fast, sensitive impedimetric DNA detection. Analyst 2012;137(1):59-63 67.

Corrigan D, Schulze H, Henihan G, et al. Impedimetric detection of single-stranded PCR products derived from methicillin resistant Staphylococcus aureus (MRSA) isolates. Biosens Bioelectron 2012;34(1): 178-84

68.

Luo C, Tang H, Cheng W, et al. A sensitive electrochemical DNA biosensor for specific detection of Enterobacteriaceae bacteria by Exonuclease III-assisted signal amplification. Biosens Bioelectron 2013;48: 132-7

69.

Tudos A, Schasfoort R. Introduction to surface plasmon resonance. In: Handbook of surface plasmon resonance. 2008. p. 1-14

70.

Cooper M. Label-free screening of bio-molecular interactions. Anal Bioanal Chem 2003;377(5):834-42

71.

Wang J, Luo Y, Zhang B, et al. Rapid label-free identification of mixed bacterial infections by surface plasmon resonance. J Transl Med 2011;9(1):85

72.

Nelson BP, Grimsrud T, Liles M, et al. Surface plasmon resonance imaging measurements of DNA and RNA hybridization adsorption onto DNA microarrays. Anal Chem 2001;73(1): 1-7

73.

Bailey R, Washburn A, Qavi A, et al. A robust silicon photonic platform for multiparameter biological analysis. In: Proceedings of SPIE. 2009. p. 72200N-72200N–6

74.

Genalyte, Inc. Available from: www. genalyte.com

63.

McKendry R, Zhang J, Arntz Y, et al. Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array. Proc Natl Acad Sci USA 2002;99(15):9783-8

75.

Scheler O, Kindt J, Qavi A, et al. Labelfree, multiplexed detection of bacterial tmRNA using silicon photonic microring resonators. Biosens Bioelectron 2012;36(1): 56-61

64.

Palchetti I, Mascini M. Amperometric biosensors for pathogenic bacteria detection. In: Zourob M, Elwary S, Turner A, editors. Principles of Bacterial Detection: Biosensors, Recognition Receptors and Microsystems. Springer, New York, NY, US; 2008. p. 299-312

76.

Woo P, Lau S, Teng J, et al. Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories. Clin Microbiol Infect 2008; 14(10):908-34

77.

65.

Wu J, Chumbimuni-Torres K, Galik M, et al. Potentiometric detection of DNA hybridization using enzyme-induced metallization and a silver ion selective electrode. Anal Chem 2009;81(24): 10007-12

Guschin D, Mobarry B, Proudnikov D, et al. Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology. Appl Environ Microbiol 1997;63(6):2397-402

78.

Salipante S, Sengupta D, Hoogestraat D, et al. Molecular Diagnosis of Actinomadura madurae infection by 16S rRNA Deep

66.

Kaatz M, Schulze H, Ciani I, et al. Alkaline phosphatase enzymatic signal amplification



Sequencing. J Clin Microbiol 2013;51(12): 4262-5 •


79.

80.

•

81.

82.

Recent case report describing correct diagnosis of pathogenic infection using next-generation sequencing when culture and conventional molecular methods did not deliver.

100.

Lee MJ, Jang SJ, Li XM, et al. Comparison of rpoB gene sequencing, 16S rRNA gene sequencing, gyrB multiplex PCR, and the VITEK2 system for identification of Acinetobacter clinical isolates. Diagn Microbiol Infect Dis 2014.78(1):29-34

101.

Zhou G, Wen S, Liu Y, et al. Development of a DNA microarray for detection and identification of Legionella pneumophila and ten other pathogens in drinking water. Int J Food Microbiol 2011;145(1): 293-300

Glazunova O, Raoult D, Roux V. Partial sequence comparison of the rpoB, sodA, groEL and gyrB genes within the genus Streptococcus. Int J Syst Evol Microbiol 2009;59(Pt 9):2317-22

Garcıá-Martıńez J, Besco´s I, Rodrı´guez-Sala J, Rodrı´guez-Valera F. RISSC: a novel database for ribosomal 16S-23S RNA genes spacer regions. Nucleic Acids Res 2001;29(1):178-80

102.

92.

Lenz O, Beran P, Fousek J, Mra´z I. A microarray for screening the variability of 16S-23S rRNA internal transcribed spacer in Pseudomonas syringae. J Microbiol Methods 2010;82(1):90-4

Peterson G, Bai J, Nagaraja T, Narayanan S. Diagnostic microarray for human and animal bacterial diseases and their virulence and antimicrobial resistance genes. J Microbiol Methods 2010;80(3):223-30

103.

93.

Keiler K, Shapiro L, Williams K. tmRNAs that encode proteolysis-inducing tags are found in all known bacterial genomes: a two-piece tmRNA functions in Caulobacter. Proc Natl Acad Sci USA 2000; 97(14):7778-83

Strommenger B, Schmidt C, Werner G, et al. DNA microarray for the detection of therapeutically relevant antibiotic resistance determinants in clinical isolates of Staphylococcus aureus. Mol Cell Probes 2007;21(3):161-70

104.

94.

Lee S, Bailey S, Apirion D. Small stable RNAs from Escherichia coli: evidence for the existence of new molecules and for a new ribonucleoprotein particle containing 6S RNA. J Bacteriol 1978;133(2):1015-23

Kim H-J, Park S-H, Lee T-H, et al. Microarray detection of food-borne pathogens using specific probes prepared by comparative genomics. Biosens Bioelectron 2008;24(2):238-46

105.

95.

Glynn B, Lacey K, Reilley J, et al. Quantification of Bacterial tmRNA using in vitro Transcribed RNA Standards and Two-Step qRT-PCR. Res J Biol Sci 2007; 2(5):564-70

Calistri A, Salata C, Parolin C, Palu` G. New generation sequencing in pathogen discovery and microbial surveillance. Expert Rev Anti Infect Ther 2013;11(9): 877-9

106.

96.

Mignard S, Flandrois J-P. Identification of Mycobacterium using the EF-Tu encoding (tuf) gene and the tmRNA encoding (ssrA) gene. J Med Microbiol 2007;56(Pt 8): 1033-41

Boers S, van der Reijden W, Jansen R. High-throughput multilocus sequence typing: bringing molecular typing to the next level. PLoS One 2012;7(7):e39630

107.

Dunne W, Westblade L, Ford B. Next-generation and whole-genome sequencing in the diagnostic clinical microbiology laboratory. Eur J Clin Microbiol Infect Dis 2012;31(8):1719-26

108.

Joseph S, Read T. Bacterial population genomics and infectious disease diagnostics. Trends Biotechnol 2010;28(12):611-18

109.

Sherry N, Porter J, Seemann T, et al. Outbreak investigation using high-throughput genome sequencing within a diagnostic microbiology laboratory. J Clin Microbiol 2013;51(5):1396-401

•

A good example of outbreak investigation using whole-genome next-generation sequencing in clinical laboratory.

110.

Shallom S, Weeks J, Galindo C, et al. A species independent universal bio-detection microarray for pathogen forensics and phylogenetic classification of

This article nicely demonstrates the discriminative power of alternative target regions over 16S rRNA in certain bacterial genus. Yamamoto S, Harayama S. PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of pseudomonas putida strains. Appl Environ Microbiol 1995;61(10):3768 Weng F, Chiou C, Lin P, Yang S. Application of recA and rpoB sequence analysis on phylogeny and molecular identification of Geobacillus species. J Appl Microbiol 2009;107(2):452-64

84.

Mollet C, Drancourt M, Raoult D. rpoB sequence analysis as a novel basis for bacterial identification. Mol Microbiol 1997;26(5):1005-11

85.

Martens M, Weidner S, Linke B, et al. A prototype taxonomic microarray targeting the rpsA housekeeping gene permits species identification within the rhizobial genus Ensifer. Syst Appl Microbiol 2007;30(5): 390-400

88.

Gu¨rtler V. The role of recombination and mutation in 16S-23S rDNA spacer rearrangements. Gene 1999;238(1): 241-52

Available from: http://linkinghub.elsevier. com/retrieve/pii/S0167701203000654

91.

Scho¨nhuber W, Le Bourhis G, Tremblay J, et al. Utilization of tmRNA sequences for bacterial identification. BMC Microbiol 2001;1:20

87.

90.

Milyutina I, Bobrova V, Matveeva E, et al. Intragenomic heterogeneity of the 16S rRNA-23S rRNA internal transcribed spacer among Pseudomonas syringae and Pseudomonas fluorescens strains. FEMS Microbiol Lett 2004;239(1):17-23

Petti CA. Detection and identification of microorganisms by gene amplification and sequencing. Clin Infect Dis 2007;44(8): 1108-14

83.

86.

89.

Review

Case R, Boucher Y, Dahllo¨f I, et al. Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies. Appl Environ Microbiol 2007;73(1):278-88 Kilian M, Poulsen K, Blomqvist T, et al. Evolution of Streptococcus pneumoniae and its close commensal relatives. PLoS ONE 2008;3(7):e2683 Hong B-X, Jiang L-F, Hu Y-S, et al. Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections. J Microbiol Methods 2004;58(3):403-11


97.

Wang L-T, Lee F-L, Tai C-J, Kasai H. Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group. Int J Syst Evol Microbiol 2007.57(Pt 8): 1846-50

98.

Huang C-H, Chang M-T, Huang L. Use of novel species-specific PCR primers targeted to DNA gyrase subunit B (gyrB) gene for species identification of the Cronobacter sakazakii and Cronobacter dublinensis. Mol Cell Probes 2013.27(1):15-18

99.

Ko K, Hong S-K, Lee K-H, et al. Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB). J Microbiol Methods [Internet]. 2003.54(3):325-37.

499

Review


114.

BioHelix Corp. Available from: www. biohelix.com

118.

TwistDx Ltd. Available from: www.twistdx. co.uk

111.

Roche Diagnostics Corporation. Available from: www.roche-applied-science.com

115.

Eiken Chemical Co., Ltd. Available from: www.loopamp.eiken.co.jp

119.

Becton, Dickinson and Co. Available from: www.bd.com

112.

Cepheid. Available from: www.cepheid.com

116.

DiaSorin. Available from: www.diasorin.com

120.

113.

Siemens Healthcare. Available from: www. medical.siemens.com

117.

Biomerieux Clinical Diagnostics. Available from: www.biomerieux-diagnostics.com

Hologic Gen-Probe, Inc. Available from: www.gen-probe.com


unknown microorganisms. BMC Microbiol 2011;11(1):132

500


Nucleic acid aptamers: research tools in disease diagnostics and therapeutics.

Emerging Loop-Mediated Isothermal Amplification-Based Microchip and Microdevice Technologies for Nucleic Acid Detection.

Multiple nucleic acid labeling and rainbow detection.

Recent Advances in Developing Small Molecules Targeting Nucleic Acid.

Advances in nucleic acid-based detection methods.

Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica.

Editorial overview: Nucleic acids and their protein complexes: Progress in nucleic acid structural biology: new technologies and discoveries.

C2c2.

Nucleic acid detection systems for enteroviruses.

Recognition of nucleic acid junctions using triptycene-based molecules.

Nucleic acid detection in the diagnosis and prevention of schistosomiasis.

Advances in multiplex nucleic acid diagnostics for blood-borne pathogens: promises and pitfalls.

Centrifugal Microfluidic System for Nucleic Acid Ampliﬁcation and Detection.

Solar-thermal complex sample processing for nucleic acid based diagnostics in limited resource settings.

Successful Combination of Nucleic Acid Amplification Test Diagnostics and Targeted Deferred Neisseria gonorrhoeae Culture.

Nucleic acid aptamer-guided cancer therapeutics and diagnostics: the next generation of cancer medicine.

Mycoplasma pneumoniae: Current Knowledge on Nucleic Acid Amplification Techniques and Serological Diagnostics.

Current Nucleic Acid Extraction Methods and Their Implications to Point-of-Care Diagnostics.

The predictive power of synthetic nucleic acid technologies in RNA biology.

Analysis of single nucleic acid molecules in micro- and nano-fluidics.

Detection of BCR-ABL T315I mutation by peptide nucleic acid directed PCR clamping and by peptide nucleic acid FISH.

Enzyme-assisted target recycling (EATR) for nucleic acid detection.

Detection of nucleic acid hybridization using surface plasmon resonance.

Nucleic Acid-based approaches for detection of viral hepatitis.