Molecular and Cellular Probes (1990) 4, 353-365

Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water Asim K . Bej,' * Meena H. Mahbubani, 2 Richard Miller, 2 Joseph L . DiCesare,3 Lawrence Haff 3 and Ronald M . Atlas' 'Department of Biology, University of Louisville, Louisville, KY 40292, U .S.A . 2 Department of Microbiology and Immunology, University of Louisville Health Sciences Center, Louisville, KY 40292, U .S.A . and 3 Perkin Elmer Corporation, 761 Main Avenue, Norwalk, CT 06859, U .S .A . (Received 23 January 1990, Accepted 26 March 1990)

Detection of pathogens (Legionella species) and indicator bacteria (coliform bacteria) was achieved by multiplex (simultaneous) PCR amplification of diagnostic gene sequences and by hybridization to immobilized poly-dT-tailed capture probes using a dot- or slot-blot approach . Complex manipulations of primer concentrations and staggered additions of primers were required in order to achieve equal amplification of multiple genes . Multiplex PCR amplification of two different Legionella genes, one specific for L. pneumophila ( mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered amplification . Multiplex PCR amplification using differing amounts of primers specific for lacZ and lamB genes permitted the detection of coliform bacteria and those associated with human faecal contamination, including the indicator bacterial species E . coli and enteric pathogens Salmonella and Shigella . Hybridization of biotin-labelled amplified DNA, in which the biotin was incorporated during PCR amplification from biotinylated-dUTP, to immobilized 400-dT-tailed capture probes permitted specific and sensitive detection of target gene sequences . The sensitivity of colorimetric detection achieved by PCR amplification of target DNA was at a level equivalent to 1-2 bacterial cells, which is the same level of sensitivity obtained with radioactive detection. The simultaneous amplification of several genes and hybridization to immobilized capture probes with colorimetric detection is an effective, efficient and rapid detection method for various human bacterial pathogens .

KEYWORDS: polymerase chain reaction (PCR), capture probe, pathogen detection, Legionella, coliform bacteria, multiplex PCR.

*Author to whom correspondence should be addressed .

0890-8508/90/050353+13 $03 .00/0

© 1990 Academic Press Limited 353



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INTRODUCTION Several methods using the polymerase chain reaction (PCR) and gene probes have recently been described that provide the basis for the required degree of specificity and sensitivity for the detection of water-borne pathogens and indicator bacteria .` It is essential that these methods have a high reliability of specifically detecting a positive signal from the target gene that has been amplified and that a false-positive does not occur due to non-specific amplification of non-target sequences . In some applications using agarose gel electrophoresis it can be difficult to distinguish an amplified target DNA band from closely located 'ghost DNA bands' amplified from non-target sequences . Hence specific gene probes are sometimes necessary to confirm the identities of amplified DNA sequences . Recently, Saiki et al.' described a 'reverse hybridization' procedure utilizing immobilized capture probes for detection of several specific allelic mutations in human HLA-DQA DNA types . In this paper we describe the use of this approach for the detection of various bacterial pathogens and indicator species in environmental waters . This method allows the incorporation of a label during the PCR reaction and its sensitive detection by hybridization to an immobilized capture probe . To take advantage of the detection potential of immobilized capture probes, we also developed modifications of multiplex PCR, described by Chamberlain et al .6 to detect human genes, for simultaneous PCR amplification of gene sequences associated with different groups of bacteria . By using this procedure several bacterial pathogens and indicator species can potentially be detected by PCR amplification of unique genes in a single tube and by simultaneous hybridization of multiple target DNA sequences with multiple immobilized capture probes on a nylon membrane .

MATERIALS AND METHODS Bacterial DNA recovery Total genomic DNA from Escherichia colt ATCC 11775 and Legionella pneumophila (Bloomington-2, Serogroup3) was extracted by the procedure described by Ausubel et al. 7 In this procedure a 1 . 5 ml overnight culture of bacteria was lysed by 0 . 5% SDS ; DNA was treated with 0 . 7 M NaCl, polysaccharides and residual proteins were complexed with 1 o/o hexadecyltrimethyl ammonium bromide (CTAB) and removed by equal volume of phenol-chloroform-isoamyl alcohol (24 :24 :2) extractions . DNA was precipitated with 2 . 5 volumes of isopropyl alcohol, dried and resuspended in TE (10 mm Tris-HCI and 1 mm Na 2 EDTA, pH 7 . 6) buffer .

PCR amplifications of DNA PCR amplifications were performed using a GeneAmp kit with native Taq DNA polymerase and a DNA thermal cycler (Perkin-Elmer Cetus Corp ., Norwalk, CT) . For amplification of a single target DNA sequence, template DNA was initially denatured at 94°C for 1-3 min followed by . a total of 45 cycles of denaturation of the



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template DNA at 94° C for 1 min, primer reannealing at 50-60 ° C for 1 min, and extension of the primers to synthesize the complementary strands at 72°C for 1 min . Typically each PCR reaction contained 1 X PCR amplification buffer (10 X buffer contained 50 mm KCI, 100 mm Tris-HCI, pH 8 . 13, 15 mm MgCI 2 and 0 . 01 % gelatin), 200 lam of each of the dNTPs, 0 . 5-1 .M each of the primers, 10 fg to 0 . 5 gg of the genomic DNAs, 5 . 0 units Taq DNA polymerase and sterile doubled distilled water treated with 0. 1% diethyipyrocarbonate (DEPC) to achieve a total volume of 100 µl . In many cases, PCR amplified DNA was concentrated by using a Centricon 30 microconcentrator (Amicon Corporation, Danver, MA) and DNA yield was determined at 260 nm wavelength in a Lambda III spectrophotometer (Perkin-Elmer Corporation, Norwalk, CT) considering 1 unit equivalent to 50 gg double stranded DNA ml -1 . Oligonucleotide primers and probes were synthesized using a Systec DNA synthesizer (Minneapolis, MN) and purified using an Oligonucleotide Purification Cartridge (Applied Biosystems, Foster City, CA) for small samples and by HPLC using a C-8 reverse phase column (Perkin Elmer) for large samples . For detection of Legionella (all species), a 104 by segment of the coding region of the 5S rRNA gene, based on the nucleotide sequence reported by McDonell & Colwell, 8 was amplified by using two 20 mer primers L5SL9 (5'-ACTATAGCGATTTGGAACCA-3') and L5SR93 (5'-GCGATGACCTACTTTCGCAT-3'). 2 A 50 mer oligonucleotide probe L5S-1 (5'CTCGAACTCAGAAGTCAAACATTTCCGCGCCAATGATAGTGTGAGGCTTC-3') was used for the detection of the amplified 5S rRNA coding gene . 2 For detection of L. pneumophila (all serotypes), a 650 by region of the coding sequence of the macrophage infectivity potentiator (mip) gene of L. pneumophila, the nucleotide sequence of which was reported by Engleberg et al .,' was amplified by using two 21 mer primers, LmipL920 (5'-GCTACAGACAAGGATAAGTTG-3') and LmipR1548 (5'GTTTTGTATGACTTTAATTCA-3') . 2 A 50 mer oligonucleotide probe Lmip-1 (5'TTTGGGGAAGAATTTTAAAAATCAAGGCATAGATGTTAATCCGGAAGCAA-3') was used for the detection of amplified mip gene . 2 For detection of coliform bacteria, including Escherichia, Shigella and Enterobacter species, 24 mer primers ZL-1675 (5'-ATGAAAGCTGGCTACAGGAAGGCC) and ZR-2548 (5'-CACCATGCCGTGGGTTTCAATATT) were used to amplify a 875 by region of the lacZ gene and a 50 mer probe LZ-1 (5'-TGACGTCTCGTTGCTGCATAAACCGACTACACAAATCAGCGATTTCCATT) was used . for hybridizations .' For the detection of Salmonella, Shigella and E . colt, a 554 by region of the lamB gene of E . coli was amplified using two 24 mer primers, BL-4899 (5'-GGATATTTCTGGTCCTGGTGCCGG) and BR-5452 (5'-ACTTGGTGCCGTTGTCGTTATCCC) .' A 50 mer oligonucleotide probe, LB-1 (5'-T000TGATAACTATCGTCTGGTTGATGGCGCATCGAAAGACGGCTGGTTG), was used for the detection of the amplified DNA .' For dual amplification of E . coll lacZ and lamB genes, varying concentrations of the primer pairs for the lac Z and lam B genes were used . The relative concentrations of the primer pairs were varied from 0. 125-1 . 0 tM to achieve equal amplification of the two target regions . The PCR conditions for these multiplex (simultaneous) amplifications were as follows : initial denaturation for 3 min at 94°C followed by a total of 45 cycles with each cycle programmed for 94°C denaturation for 1 min, 55°C primer reannealing for 1 min, and 72°C primer extension for 1 min . For dual amplification of the mip and 5S rRNA genes of L. pneumophila, varying concentrations of the primer pairs (0. 01-0 . 5 µM) were tested . Additionally, tests were



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run with varying numbers of PCR cycles using the mip primers alone followed by adding the 5S rRNA primers for dual amplification cycles . To produce radiolabelled amplified DNA, instead of adding 200 gm dATP in the dNTP mixture, a mixture of 20 µM [a-32 P]dATP (New England Nuclear, Boston, MA) and 180 µM dATP was added to the dNTP mix containing 200 µM of each of the other nucleotides and PCR was performed . Similarly, to produce biotinylated amplified DNA, instead of adding 200 µM dTTP, a mixture of 50 µM biotin-11-dUTP (Sigma Chemicals, St. Louis, MO) and 150 µM of dTTP 10 was added to the PCR reaction along with 200 µM of the other nucleotides .

Immobilized capture probe hybridizations and detection of the amplified DNAs Homopolymer dT-tails were synthesized at the 3'-OH end of each of the oligonucleotide capture probes 11 using 100 pmols of oligonucleotide probe and 25-80 units of terminal deoxynucleotidyltransferase (TdT ; IBI, New Haven, CT) in the presence of 1 x TdT buffer (1 X TdT buffer contains 100 mm potassium cacodylate, 25 mm TrisHCI, 1 MM CoCl 2, 0. 2 mm dithiothreitol, pH 7 . 6) 12 and 10-110 nmol deoxyribonucleotide triphosphate (dTTP) (Perkin-Elmer-Cetus Inc ., Norwalk, CT) . This reaction was incubated at 37°C for 1-1 . 5 h . The reaction was stopped by addition of 10 mm Na2 EDTA . The length of the homopolymer dT-tail was controlled by limiting the dTTP and TdT amounts . For example, a 400 homopolymer dT-tail was synthesized by the addition of 110 nmol of dTTP and 80 units of TdT enzyme in the above reaction containing 100 pmol of the 50 mer synthetic oligonucleotide probe . Similarly, at the 3'-OH end of a 17 mer universal bacterial oligonucleotide probe" that detects a conserved ribosomal RNA sequence of eubacteria, a 400 poly-dT-tail was added and used as control . The poly-dT-tailed oligonucleotide probes were purified twice by using Centricon-30 microconcentrators (Amicon, Danvers, MA) in TE buffer (pH 7 . 6) . The number of dTTPs incorporated at the 3'-OH end of each of the oligonucleotide probes was determined by migration in ethidium bromide (2 X 10 -4 % solution) stained 10% polyacrylamide gels in 1 X TBE buffer (1 X TBE buffer contained, 0 . 1 M boric acid, 0 . 1 M Tris-HCI, 2 mm Na 2 EDTA, pH 7 .0) at 9 V cm -1 for 1 . 5-2 h . To immobilize the oligonucleotide probes onto nylon membranes, probes containing 50-, 150-, 300- and 400-dT-tails at their 3'-OH ends, were treated with 0 . 3 M NaOH for 5 min at room temperature, neutralized with 2 . 5 M NH 4 OAc, diluted in 6 X SSC (1 X SSC contained, 0 . 15 M NaCl, 0. 015 M trisodium citrate) and spotted on 6 X SSC soaked Zetaprobe or Hybond-N nylon membranes (BioRad, Richmond, CA or Amersham Inc ., Arlington Heights, IL, respectively) using a dot- or slot-blot manifold (BioRad) . The membranes were covered with saran wrap (Fisher Scientific, Pittsburg, PA) and exposed to a totals of 230 mJ cm-2 U .V . irradiation (230 nm wavelength) (Black Light Eastern Corp ., Long Is1an°dr,NY) with the DNA side up at a 2 inch distance . 1,14 The u .v . treated membranes were washed :n 100 ml of 6 X SSC with 0 . 5% SDS at 42 ° C for 3-5 min and were stored at 4 ° C in a sealed hybridization bag until use . To determine the crosslinking of the oligonucleotide probes containing various lengths of homopolymer dT-tail, with the nylon membrane, the probes were labelled at their 5'-ends with [y- 32 P]ATP (specific activity using > 3000 Ci



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mmol - ') (New England Nuclear Corp ., Boston, MA) and T4 polynucleotide kinase (U .S . Biochemical, Cleveland, OH) .' The nylon membrane was spotted with 3 or 6 pmols of such radiolabelled probes, u .v . irradiated, washed and the radioactivity of each spot was measured in a scintillation counter . Unwashed spots containing 5'radiolabelled oligonucleotide probes with various lengths of homopolymer poly-dTtail were used as controls (100% retention) . PCR amplified DNAs were denatured by boiling in a water bath for 10 min followed by plunging in ice for 10 min . Approximately 50 ng amplified DNA (as determined by spectrophotometric analysis) was used for hybridization . For the detection of the radiolabelled DNA, oligonucleotide probes with poly-dT-tails, fixed on the Zetaprobe membrane were treated with 7 ml of hybridization solution containing 1 mm Na 2 EDTA, 0 . 5 M NaH 2 PO 4, pH 7 . 2, and 7% SDS (BioRad) in a hybridization bag (BRL, Gaithersburg, MD) at 65°C for 5 min . Then, the buffer from the bag was discarded and an equal amount of fresh hybridization buffer was added followed by addition of radiolabelled denatured amplified DNA . Hybridization was at 50-55°C in a water bath for 2-4 h with gentle shaking . The membranes were washed twice in 2 X SSPE, 0 . 5% SDS at 42 ° C for 10 min each and once in 0 . 1 X SSPE, 0 . 1 % SDS at 55 ° C for 3-5 min with gentle agitation . The membranes were dried between two Whatman 3M filters, covered with saran wrap and a Kodak X-AR film was exposed for 0 . 5-8 h . Hybridizations were performed with fixed amounts (6 pmols) of each of the immobilized capture probes containing variable lengths of homopolymer W)-tail (50, 150, 300 and 400) and approximately 50 ng (0 . 233 pmol) of [a- 32 P]dCTP labelled amplified DNA specific for each of the capture probes . Following hybridizations the filters were washed and the hybridization spots were cut out and placed in a scintillation counter . The efficiency of hybridization for each known amount of immobilized capture probe with radiolabelled amplified target DNA was determined as follows : percent immobilized probe hybridized to target = amount of target hybridized to immobilized probe/amount of probe immobilized X 100 . For detection of biotin-labelled DNA, the poly-dT-tailed oligonucleotide probes were affixed to Hybond-N membranes . The Hybond-N membranes were treated with 10 ml of hybridization solution containing 5 X SSPE (1 X SSPE is 10 mm sodium phosphate, pH 7-0,0-18 M NaCl, 1 mm Nâ 2EDTA), 0 . 5% SDS, 5 X Denhardt's solution (1 X Denhardt's solution contained, 0 . 1 g Ficoll, 0 . 1 g polyvinylpyrrolidone and 0 . 1 g bovine serum albumin, type V in 100 ml of water),7 and 100 ltg ml - ' phenol extracted, denatured, salmon sperm DNA (Sigma) or 50 .tg ml - ' type X Baker's yeast tRNA (Sigma) in a 55°C water bath for 20-30 min with gentle agitation . The buffer was removed from the bag and an equal amount of fresh hybridization buffer was added to the membrane along with biotinylated, denatured amplified DNA and hybridization was performed in a water bath at 50-55 ° C for 2-4 h with gentle agitation . The membrane was then washed as described above for radioactive detection . The colorimetric detection of biotinylated DNA was performed by using a BlueGene kit (BRL) . In this procedure the Hybond-N nylon membrane was blocked with filter sterilized 3% bovine serum albumin (crystalline, type V) (Sigma) in buffer A (0 . 1 M Tris-HCI, pH 7 . 5, 0 . 15 M NaCI) at 65 ° C for 30-45 min in a water bath with gentle shaking . The membrane was washed once in buffer A for 2-3 min at room temperature and then incubated at room temperature in 2 gI of streptavidin-



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alkaline phosphatase (SAP) conjugate (BlueGene kit, BRL) per 10 ml of buffer A for 57 min with gentle agitation . The filter was then washed extensively in buffer A for 0. 5 h with several changes (200 ml each time) at room temperature and incubated in buffer C (0 . 1 M Tris-HCI, pH 9 . 5, 0 . 1 M NaCl, 50 MM MgCI2 ) for 10-15 min . The colour development was performed in 33 µl nitroblat tetrazolium (NBT) and 25 µl 5-brorno4-chloro-3-indolylphosphate (BCIP) (BlueGene kit, BRL) mixed in 7. 5 ml buffer C between 10-60 min sealed in a hybridization bag in the dark . The filters were washed once in 200 mm Tris-HCI, pH 7 . 5 and 0 . 5 mm Na 2 EDTA to terminate the colour development .

` RESULTS Specificity and sensitivity of immobilized capture probe for L . pneumophila And E. coli detection The enzymatic tailing of the oligonucleotide probes was controlled by varying the concentrations of the dTTP and the TdT enzyme . The extent of crosslinking to the nylon membrane of the oligonucleotide probes by u .v .-irradiation containing 50, 150, 300 and 400-dT-tails was 72%, 80%, 88% and 94%, respectively, compared to the unwashed control spot (100% retention) . The crosslinking of the oligonucleotide probes to the nylon membrane was reduced when the u .v . irradiation was decreased to less than 200 m) cm -2 . u .v . irradiation greater than 230 mJ cm -2 did not increase crosslinking . Maximum crosslinking was achieved for oligonucleotide probes with the 400-dT-tailed oligonucleotide probes at 230 mJ cm -2 u .v . irradiation . When varying concentrations (3, 0 . 3 and 0. 03 pmols) of the 400 dT-tailed immobilized capture probes, L5S-1 for Legionella detection and Lmip-1 for L . pneumophila detection and a control 400-dT-tailed universal bacterial oligonucleotide probe 13 were used, specific hybridizations were detected only with the respective amplified DNAs and no non-specific hybridizations were observed (Fig . 1) . Similarly, in the experiments performed for the detection of E . coli, IacZ amplified DNA hybridized with varying concentrations (3, 0 . 3 and 0.03 pmols) of 400-dT-tailed immobilized capture probe was specific for the target DNA and no hybridization was observed either with IamB specific immobilized capture probe, or a 400--dTtailed universal bacterial oligonucleotide probe present on the same strip of nylon membrane (Fig . 2) . When amplified DNA from IamB of E . coli was hybridized to a nylon membrane strip containing immobilized oligonucleotide probes specific for IacZ and lamB and 400-dT-tailed universal bacterial oligonucleotide probe, hybridization was observed only with the IamB oligonucleotide probe (Fig . 2) . The hybridization signals using approximately 50 ng of the amplified DNA (from 10 fg of genomic DNA) in the above experiments were strongly positive with 3 prnol and detectable with 0 . 3 pmol of the immobilized poly-dT-tailed oligonucleotide probes (Figs . 1 and 2) . Based on the estimate that 10 fg of genomic DNA is equivalent to 1-2 bacterial cells, 1 '2 in this study it was possible to detect the target DNA amplified from the equivalent of approximately 1-2 bacterial cells . When PCR amplified DNA from the mip gene, containing randomly incorporated radiolabel during amplification, was used for hybridization with 6 pmols of immobilized 400-dT-tailed mip-1 oligonucleotide probe, an intense hybridization signal was



PCR amplification and immobilized capture probes

- 3 pmol _ 5S rRNA + 400 cIT

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- 3 pmoL universal bacterial probe + poly-dT-tail - 3 pmol -0~3pmol

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- 0 . 03 pmol -3 pmol universal bacterial probe + poly-dT-tail Fig. 1 . Slot-blot colorimetric detection of L . pneumophila using immobilized capture probe hybridization . Different concentrations (3, 03, 0 .03 pmols) of 400-dT-tailed L55-1 and Lmip-1 50 mer oligonucleotide probes were immobilized on the nylon membrane and hybridized with approximately 50 ng of the amplified L . pneumophila DNA (amplified from 10 fg of genomic DNA which is equivalent to 1-2 bacterial cells) . Controls included 400-dT-tailed universal bacterial oligonucleotide probe . Strip(a)-hybridization of L. pneumophila DNA multiplex amplified with 5S rRNA and mip primers with 400-dT-tailed immobilized oligonucleotide probes. Strip(b)-hybridization of L . pneumophila DNA amplified only with 5S rRNA primers with 400-dT-tailed immobilized oligonucleotide probes . Strip(c)-hybridization of L. pneumophila DNA amplified only with mip primers with 400-dT-tailed immobilized oligonucleotide probes .

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-0 . 03 pmoL - 3 pmoL universal bacterial probe + poly-dT-tail - 3 pmoL -0 . 3 pmot

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- 0 . 03 pmot - 3 pmoL universal bacterial probe + poly-dT-tail Fig. 2 . Slot-blot colorimetric detection of E. coli using immobilized capture probe hybridization . Different concentrations (3, 03, 0. 03 pmols) of 400-dT-tailed lacZ and Iamb oligonucleotide probes were immobilized on the nylon membrane and hybridized with approximately 50 ng of the amplified E. coli DNA (amplified from 10 fg of genomic DNA which is equivalent to 1-2 bacterial cell) . Controls included 400-dT-tailed universal bacterial probe. Strip(a)-hybridization of E . coli DNA multiplex amplified with lacZ and IamB primers with 400-dT-tailed immobilized oligonucleotide probes . Strip(b)-hybridization of E. coli DNA amplified only with IamB primers with 400-dT-tailed immobilized oligonucleotide probes . Strip(c)-hybridization of E . coli DNA amplified only with lacZ primers with 400-dT-tailed immobilized oligonucleotide probes .

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observed (Fig . 3a) . Detectable hybridization signals also were observed with 0. 6 pmols and 0 . 06 pmols of the immobilized 400-dT-tailed mip-1 oligonucleotide probe, respectively (Fig . 3a) . Similar results were obtained with E . colt DNA amplified with IamB primers and hybridized with 400-dT-tailed LB-1 oligonucleotide probe (Fig . 3b) . Higher radioactive counts were detected using randomly incorporated 32 Plabelled dATP (1 . 5-2 . 8 X 1010 dpm per gg of amplified DNA) than with 5'-end labelled primers (3-9 X 104 dpm per .tg of amplified DNA) . The efficiency of hybridization using PCR amplified DNA containing randomly incorporated [a32 P]dATP label during amplification, with immobilized capture probe containing 400dT-tail, was 1 . 1-1 . 3% (Tables 1 and 2) . At most, a very weak hybridization (,< 0 . 01 %) was seen with the u .v .-fixed untailed oligonucleotide probes . Hybridization efficiency with immobilized oligonucleotide probes containing less than 400-dT-tail (0, 50, 150, 300 dt-tail) was found to be 0 . 00038-1 .3% (Table 2) . The percent target that hybridized to the immobilized capture probe increased from 0-01% to 34% as the size of the poly-dT-tail was increased from 0 to 400 (Table 2) . A maximum of 2934% of the labelled target was found to hybridize with the immobilized capture probe containing a 400-dT-tail (Tables 1 and 2) .

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- 0 . 6 pmol

- 0 . 06 pmol

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Fig. 3. (a)Slot-blot detection of (radiolabelled) L. pneumophila DNA amplified with mip primers using immobilized capture probe hybridization with different concentrations (6, 0 .6, 0. 06 and 0006 pmols) of 400-dT-tailed Lmip-1 probe. Controls included 10 ng of salmon sperm DNA and 100 gl of 1 x SSC spotted on the nylon membrane. (b)Dot-blot detection of radiolabelled E . colt DNA amplified with IamB primers using immobilized capture probe hybridization with varying concentrations (6, 0 . 6, 0 .06 and 0 .006 pmols) of 400-dT-tailed IamB probe . Controls included 10 ng of salmon sperm DNA and 100 µl of 1 x SSC spotted on the nylon membrane . In all cases, approximately 50 ng of amplified DNAs (amplified from 10fg of genomic DNA which is equivalent to 1-2 bacterial cell) were used to hybridize with immobilized capture probes .



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Simultaneous dual detection of amplified genes by multiplex amplification When multiplex PCR was performed using equal concentrations of primers for both the 1acZ and 1amB genes of E. coli only 1acZ amplification occurred . To achieve multiplex amplification of both 1acZ and /amB, the IamB primers had to be in higher concentration than the 1acZ primers (Fig . 4a) . Equal amplification was achieved using a 4 :1 ratio of IamB to 1acZ primers . Hybridizations to immobilized capture probes with simultaneously amplified lacZ and lamB DNA from an initial amount of 10 fg of genomic DNA of E . coli, and biotin-SAP colorimetric detection system showed positive hybridization signals with the respective 400-dT-tailed oligonucleotide probes and no hybridization with the 400-dT-tailed universal bacterial oligonucleotide probe (Fig . 2a) . Unlike the multiplex PCR of 1acZ and /amB, adjusting the relative concentrations of primers alone was insufficient to achieve simultaneous amplification of the 5S rRNA and mip genes of Legionella . To achieve multiplex PCR amplification of the target Legionella DNA sequences, it was necessary to initiate amplification of the mip sequence before the 5S rRNa primers were added . Simultaneous addition of both primer sets resulted in amplification of only the 5S rRNA segment . Successful multiplex amplification of the 5S rRNA and mip genes of L . pneumophila was achieved when 0 . 5 .tM of each of the mip primers was first used for amplification in seven PCR cycles-in which each cycle consisted of 94°C denaturation for 1 min, 50 ° C primer reannealing for 1 min, and 72 ° C primer extension for 1 min-

Fig. 4. Ethidium bromide stained 10% polyacrylamide and 1% Agarose gel electrophoreses analysis of bacterial DNAs amplified by multiplex PCR . (a) Lane 1-E . coil DNA amplified with 1 . 0 µnn of 1acZ and 0 .5 µnn of 1amB primers . Lane 2-E. coil DNA amplified with 1 . 0 µM of 1acZ and 025 µM of 1amB primers . Lane 3-E . coil DNA amplified with 1 . 0 µM of 1acZ and 0. 125 µnn of 1amB primers. Lane 4-Lambda DNA + Hind III size standard . (b) Lane 1-L. pneumophila amplified with 0. 5 µM of mip and 0 . 5 µM of 5S rRNA primers using seven initial PCR cycles of mip amplification alone followed by 18 cycles of multiplex PCR . Lane 2-L. pneumophila amplified with 0. 5 µM of 5S rRNA primers . Lane 3-L . pneumophila amplified with 0. 5 µM of mip primers . Lane 4-Lambda DNA + Hind III size standard .



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followed by addition of 0 . 5 µm of each of the 5S rRNA primers to the reaction for 38 cycles-in which each cycle included 94°C denaturation for 1 min, 50°C primer reannealing for 1 min, and 72 ° C primer extension for 3 min (Fig . 4b) . Dual amplification was achieved in this manner and biotinylated DNA formed by PCR was detected by using 400-dT-tailed 5S rRNA and mip oligonucleotide probes (Fig . 1 a) .

DISCUSSION Since addition of equimolar quantities of primers in the PCR reaction produced no amplification of one gene (as in the case of the mip gene of Legionella when amplified in the presence of 5S rRNA primers) or unequal amplification (as in the case of the IacZ gene of E. coil when coamplified with lamB primers), two different approaches were needed to establish successful multiplex PCR, i .e., to achieve equal or near equal production of amplified DNA . In the case of coliform bacteria, adjusting the relative concentrations of primers permitted multiplex PCR amplification . In the case of multiplex amplification of Legionella genes, staggered amplifications were necessary because addition of unequal amounts of primers did not result in efficient and equal amplifications of multiple targets . The reason that a 4 :1 ratio of IamB :lacZ primers is necessary is not clear . It likewise is not known why multiplex PCR based upon adjusting the ratios of primers for Legionella 5S rRNA and mip amplification was unsuccessful, whereas initiating mip amplification for a few cycles prior to multiplex PCR worked . With regard to capture probe detection of PCR amplified DNA the colorimetric detection scheme using immobilized capture probes was performed with Hybond-N (Amersham) nylon membranes to decrease the background colour development . Similarly, we found that with radioactive detection, Zetaprobe (Biorad) nylon membrane showed minimum or no background signal . Hybridization efficiency did not correlate directly with the efficiency of binding of tailed oligonucleotide probes to nylon membrane, an observation also made by Saiki et al.' who studied the efficiency of binding of poly-dT- and poly-dC-tailed oligonucleotide probes to nylon membranes . The binding efficiencies of oligonucleatide probes without poly-dT-tails and those with less than 400 dTs, to nylon membranes decreased as the length of the tail decreased . Attaching a 400-dT-tail at the 3'-OH end of the oligonucleotide probe improved the hybridization efficiency by increasing the binding efficiency of the oligonucleotide probe to the nylon membrane and also by increasing the distance between the nylon membrane and the oligonucleotide probe sequence, thus making the oligonucleotide probe more accessible to the complementary sequence during hybridization . u .v . irradiation of 230 mJ cm -2 with an oligonucleotide probe having a 400-dT-tail gave maximum hybridization signal . This observation is consistent with those of Saiki et al .' who found that poly-dC- and poly-dTtails synthesized at the 3'-OH end of the oligonucleotide probe and attached to the nylon membrane by u .v . irradiation increased hybridization efficiency . This study indicates that by using PCR amplification with biotin labelling and immobilized capture probe hybridization methods it was possible to detect target DNA at a level equivalent to approximately 1-2 bacterial cells . Crosslinking of the oligonucleotide probes to the nylon membrane increased with the length of the



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poly-dT-tail and amount of u .v . irradiation used for immobilization, an observation that closely corresponds with the study of Saiki et a1 . 5 Although certain hybridization parameters were changed, such as, (1) longer hybridization incubation time ; (2) the use of longer probes (50 mer vs 19 mer) and 100% complementarity of the probes with the target DNA, all of which contribute to hybrid stability, the peak efficiency of hybridization of the immobilized 400-dT-tailed probes to the labelled target was equivalent to that achieved by Saiki et al . (1989) . The high sensitivity of detection was possible by use of the random incorporation of radiolabelled dATP or biotin-11-dUTP during PCR amplifications which produced very high specific activity in the amplified product . A similar finding was reported by Abott et al." Higher incorporation of biotin could be achieved by incorporating biotin-labelled nucleotides rather than using 5'-end labelled biotinylated primers during PCR amplifications, thereby reducing 10-fold the amount of biotin-labelled DNA that could be detected by using the immobilized capture probes . In conclusion, the use of multiplex PCR, incorporation of biotin during the reaction and the subsequent use of immobilized capture probe hybridization with biotinylated amplified DNA greatly reduces the time and materials required for conventional PCR-gene probe detection of pathogens and indicator bacterial species . Our results indicate that multiplex PCR and the detection of amplified DNAs by the immobilized capture probe hybridization method is specific and sensitive enough for detection of various bacterial pathogens .

ACKNOWLEDGMENTS We thank Randall Saiki and Thomas Geoghegan for helpful suggestions and constructive criticisms, Scott R . Hoffman for technical assistance and Kathy Zinn for manuscript preparation .

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Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water.

Detection of pathogens (Legionella species) and indicator bacteria (coliform bacteria) was achieved by multiplex (simultaneous) PCR amplification of d...
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