World J Microbiol Biotechnol DOI 10.1007/s11274-014-1638-8

ORIGINAL PAPER

An efficient alternative marker for specific identification of Mycobacterium tuberculosis Jianing Zhao • Yiwei Wang • Dairong Li • Jiawen Liu Xuemei Zhang • Yujuan He • Hong Wang • Ju Cao • Yibing Yin • Wenchun Xu



Received: 24 November 2013 / Accepted: 14 March 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Rapid and accurate identification of mycobacteria to the species level is important to provide epidemiological information and to guide the appropriate treatment, especially identification of the Mycobacterium tuberculosis (MTB) which is the leading pathogen causing tuberculosis. The genetic marker named as Mycobacterium tuberculosis specific sequence 90 (mtss90) was screened by a bioinformatics software and verified by a series of experiments. To test its specificity, 266 strains of microorganisms and human cells were used for the mtss90 conventional PCR method. Moreover, the efficiency of Electronic supplementary material The online version of this article (doi:10.1007/s11274-014-1638-8) contains supplementary material, which is available to authorized users. J. Zhao  X. Zhang  Y. He  H. Wang  Y. Yin  W. Xu (&) Department of Laboratory Medicine, College of Laboratory Medicine, Chongqing Medical University, 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, People’s Republic of China e-mail: [email protected] Y. Wang Department of Clinical Laboratory, Chongqing Pulmonary Hospital, Chongqing, People’s Republic of China D. Li  J. Cao Department of Pneumatology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, People’s Republic of China J. Liu Department of Clinical Laboratory, Beijing Geriatric Hospital, Beijing, People’s Republic of China Y. Yin Department of Clinical Laboratory, Children’s Hospital, Chongqing Medical University, Chongqing, People’s Republic of China

mtss90 was evaluated by comparing 16S rDNA (Mycobacterium genus-specific), IS6110 (specific identification of MTB complex), mtp40 (MTB-specific) and PNB/TCH method (traditional bacteriology testing) in Mycobacterium strains. All MTB isolates were mtss90 positive. No amplification was observed from any other tested strains with M. microti as an exception. Compared with the traditional PNB/TCH method, the coincidence rate was 99.1 % (233/235). All of the mtss90 positive strains were IS6110 and 16S rDNA positive, indicating a 100 % coincidence rate (216/216) between mtss90 and these two genetic markers. Additionally, mtss90 had a better specificity than mtp40 in the identification of MTB. Lastly, a real-time PCR diagnostic assay was developed for the rapid identification of MTB. In conclusion, mtss90 may be an efficient alternative marker for species-specific identification of MTB and could be used for the diagnosis of tuberculosis combined with other genetic markers. Keywords Molecular marker  Mycobacterium tuberculosis  PCR  Real-time PCR

Introduction Although human have struggled with tuberculosis (TB) for centuries, it remains a major global health problem. Based on the latest estimates in the Global Tuberculosis Report 2013 from WHO, it causes ill-health among millions of people every year and ranks as the second leading cause of death from an infectious disease worldwide, after the human immune deficiency virus (HIV). There were almost 8.6 million new cases and 1.3 million TB deaths in 2012 [World Health Organization, global tuberculosis report 2013 (http://www.who.int/tb/publications/global_report/

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gtbr13_main_text.pdf?ua=1). Therefore, in addition to improved therapy and new vaccines, rapid and accurate diagnosis is still urgently required for effective TB control. TB in humans is mainly caused by members of the Mycobacterium tuberculosis complex (MTBC), and also caused by nontuberculous mycobacteria (NTM). They differ widely in terms of their host tropisms, pathogenicities and therapy, so it is important to distinguish them from each other (Pinsky and Banaei 2008; Brosch et al. 2002; Sreevatsan et al. 1997; Niemann et al. 2000). In particular, as one member of MTBC, MTB is the most common pathogen in humans, accounting for more than 90 % causative agents of TB (Parsons et al. 2002). Therefore, a rapid and accurate identification of MTB is necessary and significant for the diagnosis and epidemiology of TB. The conventional identification of MTB relies on culture in combination with biochemical characteristics, which is time-consuming (nearly two months) and labor-intensive. Nucleic acid diagnostic (NAD) techniques offer rapid, reliable, and highly sensitive alternative tools for the detection of MTB (Tang et al. 2004; Yang and Rothman 2004; Jordan and Durso 2005; Qin et al. 2010). Nevertheless, the high similarity of nucleotide sequences among members of MTBC makes the species differentiation challenging (Brosch et al. 2002; Kirschner et al. 1993; Frothingham et al. 1994). The DNA coding for 16S rRNA (16S rDNA) sequence analysis (Muldrew et al. 2005; Qian et al. 2001) could be used to identify mycobacteria to species level, while it could not be applied to distinguish the members of MTBC because of the identical gene sequence (Brosch et al. 2002; Frothingham et al. 1994). The insertion sequence IS6110 has been widely used for detecting MTBC or MTB because of its high sensitivity, however some MTB strains cannot be identified for lack of IS6110 (Hellyer et al. 1996; Maurya et al. 2012; Das et al. 1995). The mtp40 marker was considered as a specific DNA marker for MTB, but it is unfortunately found to be absent in some strains of MTB and also present in M. africanum, M. canettii and some strains of M. microti (Lie´bana et al. 1996; Viana-Niero et al. 2004). In recent years, a commercial kit based on a DNA strip assay for the combined analysis of gyrB gene SNPs, 23S rRNA, and the RD1 deletion, named GenoType MTBC (Hain Lifescience GmbH, Nehren, Germany), has become available (Neonakis et al. 2007; Richter et al. 2003; Somoskovi et al. 2008). This line probe assay enables identification of the presence of various members of the MTBC but does not differentiate M. canettii from MTB. Here we report a new DNA marker for identification of MTB. It was found by the ssGeneFinder (a bioinformatics software), and then the specificity of this genetic marker was evaluated using a series of reference strains and clinical isolates by conventional PCR method. Moreover, its

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excellent validity was demonstrated by comparison with other markers. Based on these, a real-time (TaqMan) PCR assay was established for the rapid identification of MTB.

Materials and methods A total of 266 strains of microorganisms (216 clinical MTB isolates, 18 clinical NTM isolates, 16 non-Mycobacterium bacteria, 13 standard mycobacteria, 2 fungi and a Mycobacterium bovis BCG isolate) and human umbilical vein endothelial cells (HUVEC, purchased from the American Type Culture Collection) were used in this study (Table 1). The strains (inactivated) were kindly provided by the National Institutes for Food and Drug Control (NIFDC), pathogenic microbiology department of Chongqing Medical University (PMDCMU), Chongqing Pulmonary Hospital (CPH), the First Affiliated Hospital of Chongqing Medical University (FAHCMU), Children’s Hospital of Chongqing Medical University (CHCMU), and Beijing Geriatric Hospital (BGH). All clinical isolates were identified by traditional bacteriological examinations and biochemical tests. MTB isolates were identified by the PNB/ TCH (para-nitrobenzoic acid/thiophene-2-carboxylic acid hydrazide) test (Giampaglia et al. 2005), in which the growth of M. bovis is inhibited by PNB and TCH both, MTB is inhibited by PNB only and NTM is resistant to both. DNA extraction of the non-Mycobacterium bacteria was performed with corresponding medium (solid or liquid) by use of a TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China). DNA extraction of fungi was performed from Sabouraud medium by use of a E.Z.N.A.Ò Fungal DNA Kit (Omega Biotech, Doraville, USA). The genomic DNA of HUVEC was extracted by TIANamp Genomic DNA Kit (TIANGEN) from 107 cells. Samples of mycobacteria were prepared by simple thermolysis (Bouakaze et al. 2010) as follows:a loop of culture (i.e., isolates of bacteria grown on Lowenstein–Jensen medium) was suspended in 100–150 ll sterile water, and the mixture was heated at 100 °C for 15 min. The mycobacterial cells were disrupted by heat shock treatment (1 min at 95 °C, followed by 1 min on ice, repeated three times). The supernatant obtained after centrifugation at 13,000g for 2 min contained the mycobacterial DNA. All the DNA extracts were stored at -20 °C and prepared as templates for the PCR amplification. ssGeneFinder (http://147.8.74.24/ssGeneFinder/) is a novel pangenomic analysis software which allows the user to generate specific targets for any publicly available genome (Ho et al. 2011a, b, 2012). In this study, MTB was selected as the screening target. The detailed process is as follows: 57 MTB genomes were selected as targets and 83 genomes were selected as Non-target genomes (Online

World J Microbiol Biotechnol Table 1 List of microorganisms used in this study Organism

Strain number

Table 1 continued Strain source

Non-Mycobacterium bacteria

Organism NTM isolates

Gram-positive cocci

Strain number 5 1

Streptococcus pneumonia

1

ATCC49619

Staphylococcus aureus

1

ATCC25923

1

CHCMU

Bacillus cereus

1

PMDCMU

Bacillus subtilis

1

PMDCMU

Corynebacterium diphtheriae

1

PMDCMU

Clostridium tetani Clostridium perfringens

1 1

PMDCMU PMDCMU

Clostridium butyricum

1

PMDCMU

Escherichia coli

1

ATCC25922

Pseudomonas aeruginosa

1

ATCC27853

Acinetobacter baumannii

1

CHCMU

Haemophilus influenzae

1

CHCMU

Salmonella typhi

1

PMDCMU

Shigella dysenteriae

1

PMDCMU

Proteus vulgaris

1

PMDCMU

Cryptococcus neoformans

1

PMDCMU

Candida albicans

1

CHCMU

Mycobacterium tuberculosis H37Rv

1

ATCC27294

Mycobacterium avium

1

CMCCd95001

Mycobacterium intracellulare

1

CMCC95002

Mycobacterium xenopi

1

CMCC95003

Mycobacterium gastri

1

CMCC95006

Mycobacterium shimoidei

1

CMCC95008

Mycobacterium triviale

1

CMCC95009

Mycobacterium kansasii

1

CMCC95013

Mycobacterium smegmatis

1

CMCC95023

Mycobacterium microti

1

CMCC95048

Mycobacterium africanum

1

CMCC95049

Mycobacterium bovis Mycobacterium bovis BCG Denmark

1 1

CMCC95055 PMDCMU

12

Strain source CPH CHCMU BGH

ATCC cultures obtained from the American Type Culture Collection

Gram-negative cocci Neisseria meningitides Gram-positive bacilli

Gram-negative bacilli

Fungi

Mycobacteria Reference strains

Clinical isolates MTB isolates

Mycobacterium bovis BCG isolate

11

CHCMU

116

CPH

89

BGH

1

CHCMU

Resource 1). Default settings on the server were used for the analysis (E value for conserved targets, 1 9 10-40; E value for elimination, 1.0; minimum target sequence length, 50 bp; minimum percentage of genomes in which the targets must be conserved, 95 %). The sequence was obtained using the link provided in the notification email and then compared to those deposited in the GenBank database using the basic local alignment search tool on the internet (http://www.ncbi.nlm.nih.gov/BLAST/). In this study, a DNA fragment specific to MTB was found by the use of ssGeneFinder, and was named as mtss (Mycobacterium tuberculosis specific sequence). For the online BLASTn search, mtss still had no similarity to any sequence of human and non-MTB species (E value for elimination, 1.0). The target gene names, primer names, primer sequences and amplification product sizes are listed in Table 2. All of the primers used for conventional PCR were synthesized and purified by Shanghai Sangon. A series of primer pairs designed for the obtained sequence were created by using the Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, CA, USA) and the specificity was checked using a Primer-BLAST query in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/). All strains or isolates listed in the Table 1 were amplified for mtss90, while genetic markers (16S rDNA, IS6110 and mtp40) were performed in mycobacteria. The PCR reaction mixtures (50 ll) of mtss90 consisted of 5 ll 109PCR buffer (TaKaRa, Dalian, China), 1.5 mM MgCl2, 0.2 mM deoxyribonucleotide triphosphate (dNTP), 0.2 lM each primer (Mtss1, Mtss2), 2.5 U Taq DNA polymerase and 10–100 ng of template DNA. The reaction program was as follows: 94 °C for 5 min, followed by 30 cycles of 30 s at 94 °C, 30 s at 63 °C, and 15 s at 72 °C, and finally 72 °C for 5 min. The PCR reaction mixtures and programs of 16S rDNA (Kox et al. 1995), IS6110 (Maurya et al. 2012), mtp40 (Del Portillo et al. 1991), RD1 (Parsons et al. 2002), IS15610 (Huard et al. 2003), Rv1510 (Huard et al. 2003), Rv1970 (Huard et al. 2003), Rv3877/8 (Huard et al. 2003) and Rv3120 (Huard et al. 2003) were performed according to the references with some slight modification. PCR

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World J Microbiol Biotechnol Table 2 Primers used in this study Primers name

Sequence (50 –30 )

Amplicon length (bp)

Reference

mtss90

Mtss1

AGTCTCTTATTGCGCGTTTATTGCG

90

Designed by ourselves

16S rDNA

Mtss2 P1

TCGGATGCGACCGAATTTGC GAGATACTCGAGTGGCGAAC

212

Kox et al. (1995)

P2

GGCCGGCTACCCGTCGTC 123

Maurya et al. (2012)

396

Del Portillo et al. (1991)

Region present, 150 Region absent, 200

Parsons et al. 2002

943

Huard et al. (2003)

1,033

Huard et al. (2003)

1,116

Huard et al. (2003)

999

Huard et al. (2003)

404

Huard et al. (2003)

461

Designed by ourselves

About 550

Roth et al. (1998)

About 500

Roth et al. (1998)

84

Designed by ourselves

Assay type and target name PCR

IS6110 mtp40

RD1 (multiplex PCR)

IS 15610 Rv1510 Rv1970 Rv3877/8 Rv3120

IS1

CCTGCGAGCGTAGGCGTCGG

IS2

CTCGTCCAGCGCCGCTTCGG

PT1

CGGCAACGCGCCGTCGGTGG

PT2

CCCCCCACGGCACCGCCCGG

ET1

AAGCGGTTGCCGCCGACCGACC

ET2

CTGGCTATATTCCTGGGCCCGG

ET3

GAGGCGATCTGGCGGTTTGGGG

IS1561F

GCTGGGTGGGCCCTGGAATACGTGAACTCT

IS1561R

AACTGCTCACCCTGGCCACCACCATTGACT

Rv1510F

GTGCGCTCCACCCAAATAGTTGC

Rv1510R

TGTCGACCTGGGGCACAAATCAGTC

Rv1970F

GCGCAGCTGCCGGATGTCAAC

Rv1970R

CGCCGGCAGCCTCACGAAATG

Rv3877/8F

CGACGGGTCTGACGGCCAAACTCATC

Rv3877/8R

CTTGCTCGGTGGCCGGTTTTTCAGC

Rv3120F

GTCGGCGATAGACCATGAGTCCGTCTCCAT

Rv3120R

GCGAAAAGTGGGCGGATGCCAGAATAGT

F461

S1

AAACCGCAGGTCAATCGTGG

S2

ACGCTGCTTCAGCCATCCTT

16S rDNA

KY18

CACATGCAAGTCGAACGGAAAGG

KY75

GCCCGTATCGCCCGCACGCT

ITS-F

TTGTACACACCGCCCGTCA

ITS-R

TCTCGATGCCAAGGCATCCACC

TqMn-F

AGTCTCTTATTGCGCGTTTATTGC

TqMn-R Probe (FAMMGB)

GCGACCGAATTTGCGTCTCT CAAACCGGCAAGCGTC

Sequencing

ITS Real-time PCR mtss90

amplifications were performed in T100TM Thermal Cycler (Bio-Rad, Singapore). Each PCR amplification batch included a positive control for the target locus, as well as a negative control. Five microliter of each amplified products were electrophoresed in a 2–2.5 % (wt/vol) agarose gel added goldview, with a molecular size marker (TaKaRa) in parallel. Each PCR experiment was repeated at least three times.

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To evaluate the sensitivity of the conventional PCR, the standard recombinant plasmids were created in our laboratory. Based on the M. tuberculosis H37Rv complete genome, a 461 bp fragment (F461) containing the mtss90 was chosen as the target to be transformed. The primers S1 and S2 (Table 2) were assigned according to the sequence of F461. The same PCR reaction mixtures and program of mtss90 (modification of an annealing for 30 s at 58 °C and

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an extension for 40 s at 72 °C) were used for the F461 amplification. The amplicon of F461 containing the mtss90 from M. tuberculosis H37Rv was purified using TaKaRa MiniBEST DNA Fragment Purification Kit (TaKaRa), subcloned into a pMD19-T vector with a TA cloning kit (TaKaRa), transformed into E. coli DH5a strains, and sequenced (BGI Tech, Shenzhen, China). The clones with correct sequences were extracted as standard recombinant plasmids using Plasmid Mini Kit I (Omega). The samples (2 ll) of the seven serial sets of the tenfold dilutions of the prepared plasmids (107–101 copies/ll) were used as the standard templates. The target mtss90 was amplified and electrophoresed in the same way as above. The M. tuberculosis H37Rv and 30 clinical isolates were randomly selected for sequencing. Mtss90 is too short to be sequenced, while the clone and sequencing was time-consuming and inconvenient. Therefore, the F461 fragment was employed for sequencing. The F461 PCR products were amplified and sent for sequencing (BGI Tech, Shenzhen, China). But if an isolate failed to amplify F461, its 90 bp mtss90 would be subcloned into pMD19-T vector for sequencing. Moreover, the amplification products of 16S rDNA (KY18 and KY75), ITS (ITS-F, ITS-R) from 18 NTM clinical isolates were also sent for sequencing (Roth et al. 1998), and then the sequencing results were deposited in Ribosomal Differentiation of Medical Microorganisms (RIDOM, http://www.ridom-rdna.de/) for the final identification. A 99 % match with the sequence of the prototype strain in a repository was used as the criterion for species, group or complex level identification (Yam et al. 2006). A real-time (TaqMan) PCR assay was established for the rapid identification of MTB. A new pair of primers (TqMn-F, TqMn-R) and a 22-nucleotide TaqMan probe (Life Technologies, Carlsbad, CA, USA) which was labeled with the fluorescent reporter 6-carboxyfluorescein (FAM) and NFQ-MGB specific for mtss90 were designed (Table 2). The reaction mixtures (20 ll) consisted of 10 ll TaqMan Universal PCR Master Mix, 0.9 lM of each primer (TqMn-F and TqMn-R), 0.25 lM TaqMan probe (TqMn-FAM), and 2 ll templates (standard plasmids or DNA extracts of tested strains). This preparation was subjected to the program below: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and all of the amplification steps were carried out in Rotor-Gene 6000 (Corbett Life Science, Australia). To evaluate the sensitivity of the real-time PCR, the standard recombinant plasmids mentioned above were used in this part too. The samples (2 ll) of the seven serial sets of the tenfold dilutions of the prepared plasmids (107–101 copies/ll) were used as the standard templates and the standard curve for the quantitative analysis was constructed automatically by plotting each of the threshold cycle numbers (CT) values against the log of the copy numbers

for the standard templates. Moreover, the specificity of the Taqman PCR assay was evaluated with the M. tuberculosis H37Rv reference strain, randomly chosen ten clinical MTB isolates, 30 non-MTB strains (12 reference strains and 18 NTM clinical isolates shown in the Table 1), S. pneumonia, E. coli and C. albicans. The copy number of the examined samples was calculated by the standard curves.

Results Based on the MTB specific fragment mtss, a series of primer pairs were designed to identify M. tuberculosis (date not shown). A 90 bp fragment named Mycobacterium tuberculosis specific sequence 90 (mtss90) was obtained by the primer pair Mtss1 and Mtss2 which showed the best specificity. The specificity of mtss90 was studied in mycobacteria, non-mycobacterial bacterial species, fungi and HUVEC. The amplified products were only positive in the MTB genome except for M. microti (part of the results shown in Fig. 1). The PCR products from F461 amplified from M. microti were sequenced and the results showed that it was identical to mtss90 standard sequence. The amplification of standard recombinant plasmids containing the mtss90 was successfully obtained. The samples (2 ll) of the seven series of the prepared standard recombinant plasmids (107–101 copies/ll) were used as the standard templates for mtss90 amplification. The electrophoresis results were shown in the Fig. 2, and the limit for the visualization of the amplified products on the gel was 104 copies/ll. Compared with the traditional PNB/TCH method, the coincidence rate was 99.1 % (233/235, Table 3). The inconsistent two isolates were the clinical isolates named w214 (Chongqing) and 2047 (Beijing). The w214 isolate was identified as MTB by traditional PNB/TCH method, but non-MTB by the mtss90 PCR amplification. The patient’s information showed that the w214 isolate was separated from the exudates of a child who had a history of vaccine inoculation and was highly suspected of BCG (bacille Calmette-Gue´rin) infection based on the clinical manifestations. To prove our suspicion, the RD1 genetic marker and its primers (ET1, ET2, ET3) were used for a multiplex PCR to differentiate BCG strain from other MTBC species (Parsons et al. 2002; Talbot et al. 1997). A BCG strain should yield a single 200-bp product with primers ET1 and ET3 indicating deletion of the RD1 sequence, whereas a MTB strain should yield a single 150-bp product with primers ET2 and ET3 indicating that at least a portion of RD1 was present. The W214 isolate yielded a 200-bp product. Combined with the sequencing result of 16S rDNA, the W214 isolate was identified as M. bovis BCG rather than MTB. The other inconsistent 2047

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Fig. 1 Agarose gel electrophoresis showing the amplification products of mtss90 DNA fragment with primer pair Mtss1 and Mtss2. M: molecular marker GeneRuler 500-bp DNA ladder (500 bp, 400 bp, 300 bp, 200 bp, 150 bp, 100 bp, 50 bp); lane 1: S. pneumonia; lane 2: E. coli; lane 3: C. albicans; lane 4: M. avium; lane 5: M. africanum; lane 6: BCG; lane 7: M. bovis; lane 8: HUVEC; lane 9: M. microti; lane 10: M. tuberculosis H37Rv; lane 11– 16: clinical isolates of MTB; N: negative control

and a M. nonchromogeicum by the sequencing of 16S rDNA (KY18, KY75) and ITS (ITS-F, ITS-R). The standard recombinant plasmids (107–101 copies/ll) were well amplified with the quantitative PCR and the standard curve that was constructed based on CT value data collected from the TaqMan PCR is shown in Fig. 3. The standard curve demonstrated a significant linear relationship (R2 = 0.994, efficiency = 100.2 %) between the CT values and the log of the copy numbers for each of the standard plasmids. The limit of detection of this assay was 101 copies/ll. There was no amplicon in the non-MTB strains except M. microti, and the copy numbers of MTB strains measured ranged from 107 to 104 copies/ll.

Discussion

Fig. 2 The sensitivity of conventional PCR. M: molecular marker GeneRuler 500-bp DNA ladder; lane 1–7: 107–101copies/ll; N: negative control

isolate was identified as NTM by traditional PNB/TCH method, while the mtss90 amplification showed it was a MTB isolate. Moreover, the 2047 isolate was also IS6110 and mtp40 positive, indicating it was one of the MTBC but not NTM. Therefore, five genetic markers (IS15610 , Rv1510, Rv1970, Rv3877/8, and Rv3120) for PCR amplification (Huard et al. 2003) and 16S rDNA (KY18, KY75) for sequencing were used to further identify the isolate 2047. The 16S rDNA sequencing result showed it was one of the MTBC. Five primer pairs were all amplified positive, indicating it was a MTB isolate. The results above indicated an excellent specificity of mtss90 for MTB identification. All of the mtss90 positive isolates were IS6110 (MTBCspecific) and 16S rDNA (Mycobacterium genus-specific) positive, indicating a 100 % coincidence rate (216/216) between mtss90 and these two genetic markers. Mtp40 (MTB-specific) failed to identify the clinical isolate 556 which was identified as MTB by conventional test, but mtss90 was able to identify it (Table 3). Moreover, mtp40 was not able to distinguish MTB from M. africanum because it was positive in both strains, while mtss90 was able to identify it. The sequencing results from the randomly chosen 30 clinical MTB isolates were completely corresponding with the standard sequence of mtss90, indicating there was no false positive amplification. As is shown in Table 3, the 18 NTM clinical isolates were further identified as 7 M. intracellulare, 6 M. abscessus, 2 M. kansasii, 2 M. fortuitum

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MTB is the most common and important pathogen in the genus Mycobacterium. The rapid identification of MTB lays the foundations for the diagnosis, therapy, and further research of TB. However, the conventional methods for identifying MTB are time-consuming and labor-intensive. Although some genetic markers can be used to identify MTB, they still have some shortages. Follow-up restriction enzymes analysis, sequencing or combined analysis with other markers are needed because of low specificity. Here a novel DNA target mtss90 may provide a new choice for rapid and accurate identification of MTB. This target could identify MTB by simple PCR amplification without relying on follow-up restriction enzymes analysis and sequencing. The novel DNA fragment mtss specific to MTB was identified by bioinformatics software. The availability of high-throughput DNA sequencing technologies has ignited an explosion of genome data. This wealth of information provides a new approach to the selection of molecular targets. Based on the blast results in the GenBank database, mtss is present in all genomes of different genotypes of MTB in the GenBank database and shows no similarity to complete genomes of M. africanum and M. canettii. Therefore, mtss shows better specificity when compared with the mtp40 marker or the marker used in GenoType MTBC DNA strip assay (Hain LifescienceGmbH). The mtss90 assay has been evaluated in different microorganisms, including probiotic (Clostridium butyricum is commonly used as a probiotic), common pathogens, fungi, mycobacteria. As is supported by our results, mtss90 represents excellent specificity for MTB identification except for M. microti. It has been confirmed by sequencing that there was a sequence identical to the mtss90 standard sequence in the M. microti genome. However, M. microti seldom causes human infection (van Soolingen et al. 1998) which will have little influence on the clinical application of mtss90.

World J Microbiol Biotechnol Table 3 Efficiency of mtss90 in the identification of MTB

Microorganisms and cells

Source

Number

PCR results 16S rNDA

IS6110

mtp40

mtss90

MTBC MTB H37Rv

Reference

Clinical isolates

Beijing a

?

?

?

?

?

?

?

?

1

?

?

?

?

Clinical isolates

Chongqing

126

?

?

?

?

Clinical isolate 556

Chongqing

1

?

?

-

?

M. africanum

Reference

1

?

?

?

-

M. bovis

Reference

1

?

?

-

-

M. bovis BCG Denmark

Reference

1

?

?

-

-

Chongqing

1

?

?

-

-

Reference

1

?

?

?

?

M. avium

Reference

1

?

-

-

-

M. intracellulare

Reference

1

?

-

-

-

M. xenopi

Reference

1

?

-

-

-

M. gastri

Reference

1

?

-

-

-

M. shimoidei

Reference

1

?

-

-

-

M. trivial

Reference

1

?

-

-

-

M. kansasii

Reference

1

?

-

-

-

M. smegmatis

Reference

1

?

-

-

-

M. intracellulare

Beijing

7

?

-

-

-

M. abscessus isolate

Beijing

3

?

-

-

-

M. kansasii isolate

Beijing

2

?

-

-

-

M. abscessus isolate

Chongqing

3

?

-

-

-

M. fortuitum isolate M. nonchromogeicum

Chongqing Chongqing

2 1

? ?

-

-

-

Clinical isolate 2047

M. bovis BCG Clinical isolate w214b M. microti

Beijing

1 88

NTM

a

Clinical isolate 2047 was firstly identified as a NTM isolate by PNB/TCH

b

Clinical isolate w214 was firstly identified as a MTB isolate by PNB/TCH ND not done

16

ND

ND

ND

-

Fungi

Non-Mycobacterium bacteria

2

ND

ND

ND

-

HUVEC

1

ND

ND

ND

-

Fig. 3 The Standard curve of ten fold serial dilutions of standard plasmids

The efficiency of mtss90 in the identification of MTB was compared with the conventional PNB/TCH method and genetic markers 16S rDNA, IS6110 and mtp40 in 248

mycobacterial reference stains or clinical isolates. Mtss90 showed a high degree of consistency compared with the conventional PNB/TCH method or other genetic markers

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(Table 3). The conventional PNB/TCH method is time-consuming and depends too much on technicians’ subjectivity and experience which may influence the veracity and objectivity of the result. And in this study, the nucleic acid diagnostic techniques showed higher accuracy. It was worth pointing out that the two inconsistent clinical isolates. One was the clinical isolate 2047 which was isolated from an adult in Beijing. It was identified as NTM by PNB/TCH method, while mtss90 identified it as MTB. In the end, it was confirmed to be MTB by 16S rDNA sequencing and five genetic markers. The other inconsistency, w214, was isolated from a child in Chongqing with a history of BCG vaccine inoculation. It was identified as MTB by traditional PNB/TCH method, but identified as a non-MTB strain by mtss90. And it was further identified as M. bovis BCG by 16S rDNA sequencing and multiplex PCR of RD1 (Parsons et al. 2002; Talbot et al. 1997). These results indicated that mtss90 had a capacity of distinguishing MTB from BCG, and such exciting results allowed mtss90 a bright prospect in the countries where BCG vaccine has been used widely. Currently, there are some excellent genetic markers used for the molecular diagnosis of tuberculosis, but none of them is absolutely perfect. For example, the marker 16S rDNA cannot identify MTB accurately; the IS6110 has been found absent in some MTB strains; the mtp40 has been found absent in some MTB strains and also present in some strains of MTBC (e.g., M. africanum). It is worth pointing out that mtss90 showed better efficiency for MTB identification compared to mtp40. Firstly, mtss90 was amplified successfully from an mtp40 absent clinical MTB isolate 556. Secondly, the mtss90 was able to distinguish MTB from M. africanum while the mtp40 could not. In conclusion, the mtss90 showed good efficiency in MTB identification. To avoid the carryover contamination of conventional PCR (Niesters 2001; Stra´nska´ et al. 2004), a real-time PCR for mtss90 was established successfully for clinical application. The detection limit of the real-time PCR was 101 copies/ll, while the conventional PCR was 104 copies/ll. The real-time PCR showed higher sensitivity than conventional PCR. There are still some limits in our study. More kinds of mycobacteria are needed to demonstrate the specificity of mtss90. In addition, clinical samples (such as sputum, urine, pleural fluid, etc.) are needed to evaluate the availability of the target. In this study, a novel DNA marker specific to MTB was provided as a new choice for the identification of MTB. Acknowledgments We thank the National Institutes for Food and Drug Control, Guozhi Wang and Baowen Chen for their generous providing of the DNA of mycobacteria. This study was supported by the grants from Project Supported by the Scientific and Technological Research Program of Chongqing Municipal Education Commission

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(Grant No. KJ120318), the National Natural Science Foundation of China (No. 81101216), and the Natural Science Foundation of Chongqing (No. cstc2012jjA10009).

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An efficient alternative marker for specific identification of Mycobacterium tuberculosis.

Rapid and accurate identification of mycobacteria to the species level is important to provide epidemiological information and to guide the appropriat...
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