Development and Use of an Internal Positive Control for Detection of Bordetella pertussis by PCR
http://www.100md.com
微生物临床杂志 2005年第5期
Laboratoire de Bacteriologie-Hygiene, Centre Hospitalier Regional Universitaire, Lille, France
INSERM E0364, Institut de Biologie de Lille, 1 rue du Pr. Calmette, 59021 Lille Cedex, France
Faculte des Sciences Pharmaceutiques et Biologiques, Lille, France
ABSTRACT
An internal control of amplification was constructed by recombinant PCR to detect PCR inhibitors. This exogenous DNA was included in the reaction mixture and coamplified with the target gene. This detection was successfully applied to the diagnosis of whooping cough by amplification of a fragment of Bordetella pertussis IS481.
TEXT
The detection of inhibitors of amplification remains a major problem which can partly be solved by the use of internal controls (ICs) of amplification (5, 6, 19). In addition to some commercially available systems, inhibitors can be detected either by spiking negative samples with the target DNA (7), by diluting the specimen to minimize the effects of inhibitors (16), or by adding a control template (2, 10, 15).
In this study, we developed for diagnosis of Bordetella pertussis a method to detect PCR inhibitors in clinical samples. We constructed an IC that can be amplified by the same primer pair as wild-type target DNA. IC was obtained by insertion of an exogenous DNA fragment into reference strain target by recombinant PCR (1, 3, 4).
B. pertussis Tohama phase I strain was the reference strain used to amplify the repetitive insertion sequence IS481. Escherichia coli DH5 was used as host for recombinant vector pMos (Amersham Pharmacia Biotech, Raiham, England). Amplified DNA from the B. pertussis reference strain was purified with a QIAquick PCR purification kit (QIAGEN, Hilden, Germany). This wild-type fragment was then directly used as template for recombinant PCR.
A 191-bp fragment from pertussis toxin gene amplified with primers Pt1 (5'-CCAACGCGCATGCGTGCAGATTGCTC-3') and Pt2 (5'-CCCTCTGCGTTTTGATGGTGCCTATTTTA-3') was introduced into the wild-type IS481 DNA (203 bp) by recombinant PCR (Fig. 1). Two internal primers, harboring a 5' end complementarity to the extremities of the exogenous toxin gene fragment to be inserted, were designed. In a first step, two PCR fragments were independently generated: fragment A with external primer A (Bp13, 5'-CCGCGCTGTGCCATGAGCTGG-3') and internal primer A (Pt1rv/Bp15, 5'-GACGAATCTGCACGCATGCGCGTTGGGGGCCCCGCAAGGCCGACTGGATGAAGCGTTCG-3') and fragment B with external primer B (Bp14, 5'-GATGCCTTGGTGGGGTCGATG-3') and internal primer B (Pt2rv/Bp16, 5'-TAAAATAGGCACCATCAAAACGCAGAGGGGGGCCCTGAGTGGGCTTACGCTCACACCTACCA-3'). These two amplicons and the 191-bp fragment were submitted to a second step of extension without primers. The third step consisted of a PCR with Bp13 and Bp14 in order to amplify the future internal control (Fig. 1). The resulting fragment (406 bp) was cloned into pMos vector. Recombinant plasmid was designated pSIBp. Construction was confirmed by PCR on thermolysates of E. coli strains containing the recombinant plasmid. Two ApaI sites flanking the exogenous DNA were present on Pt1rv/Bp15 and Pt2rv/Bp16 and were used to check the construction by ApaI restriction of the fragment amplified from an E. coli thermolysate. Three fragments of 100, 105, and 201 bp were obtained as expected. One clone of E. coli containing the correct recombinant plasmid was kept at –80°C. pSIBp plasmid was used as IC for detection in clinical samples.
IC concentration was optimized by adding various quantities of IC to PCRs containing constant amounts of wild-type DNA. Tenfold dilutions of the pSIBp plasmid were made, from 10 ng/μl to 10–10 ng/μl, and 5 μl was added to amplification reactions containing a constant amount of bacterial DNA known to be the threshold previously determined (103 CFU/ml). A 10–7 dilution of the pSIBp was determined as the optimal amount of IC. This dilution of plasmid corresponds to 104 molecules of internal standard versus 10 copies of the bacterial genome.
At high bacterial inoculums, only the wild-type fragment was amplified and no internal standard was detected (Fig. 2). Wild-type DNA is preferentially amplified because of its shorter length and because competition is minimized by addition of a small amount of IC. Since an excess of IC or genomic DNA may inhibit the amplification of the other by competition (Fig. 2), it is necessary to optimize the quantity of IC DNA added into the reaction mixtures. The amount of IC added in each reaction tube may seem quite high (104 plasmid copies) but is in accordance with previously published results (11).
Specimens for B. pertussis detection were nasopharyngeal aspirations (NPA) routinely received by the Bacteriology Laboratory of the Lille University Medical Center, France. The NPA collection tubing was flushed out by aspiration of 1 ml of sterile distilled water. Culture and identification of B. pertussis were performed according to conventional methods (13). For PCR detection, the NPA suspensions were boiled for 15 min and then centrifuged. Five microliters of the supernatant was directly used for amplification of IS481. Reaction mixtures were subjected to 35 cycles of amplification (30 s at 94°C, 30 s at 55°C, and 60 s at 72°C) (20). Amplicons were revealed by agarose gel electrophoresis followed by ethidium bromide staining.
One hundred twenty clinical samples were used to test the reliability of the B. pertussis IS481 IC. After a first amplification with pure NPA lysates, 33 samples (27.5%) were IS481 positive and 44 specimens (36.6%) were negative for IS481 but positive for the IC; in 43 samples (35.8%) neither the IC nor IS481 were detectable, suggesting the presence of inhibitors in the NPA samples. A second PCR assay was performed on these 43 NPA lysates, which were diluted 10-fold. Three specimens were IS481 positive, 25 specimens (20.8%) were negative for IS481 but positive for the IC, and 15 (12.5%) specimens remained inhibitory to the amplification of both IS481 and the IC. Following development tests, a 10-fold dilution of the thermolysate was included in our protocol for the diagnosis of B. pertussis.
A retrospective study over 4 years on 1,188 NPA specimens to detect B. pertussis by PCR was performed. The detection of IS481 was positive in 111 cases (9.3%), whereas 200 samples (16.8%) were negative for IS481 and the IC when tested with pure lysates but positive for the IC with 10-fold-diluted samples, 821 samples were negative for IS481 but positive for the IC, and 56 samples (4.7%) were negative for both IS481 and the IC. For the latter samples, PCRs obtained with DNA extracted with a commercial kit were still negative for IS481 but positive for IC. Although some authors (9, 14) have described no inhibition phenomenon within their clinical specimens, inhibition rates from 5 to 42% are quite usual (8, 16-18), depending on the nature of the clinical samples and the DNA extraction method. The present results, established on a large number of specimens, gave a smaller number of inhibiting specimens (21.5%) than the development study (35.8%) and were in agreement with those reported in the literature.
As the IC was amplified by the same primer pair used for the detection of the microorganism, there were no major modifications of the amplification protocols. Moreover, since the diagnosis of B. pertussis by IS481 was usually confirmed by a second PCR targeting pertussis toxin, our standardized IC, combining IS481 and pertussis toxin, was used as positive control for both assays. It should be possible to improve the amplification methods by addition of the IC into the clinical specimen prior to extraction (12). This IC could allow simultaneous detection of inefficient sample preparations.
ACKNOWLEDGMENTS
We are very grateful to A. Devalckenaere and S. Armand for their technical assistance.
REFERENCES
Donnenberg, M. S., J. Yu, and J. B. Kaper. 1993. A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J. Bacteriol. 175:4670-4680.
Gibb, A. P., and S. Wong. 1998. Inhibition of PCR by agar from bacteriological transport media. J. Clin. Microbiol. 36:275-276.
Higushi, R. 1990. Recombinant PCR, p. 177-183. In M. Innis (ed.), PCR protocols: a guide to methods and applications. Academic Press, Inc., San Diego, Calif.
Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59.
Hoorfar, J., N. Cook, B. Malorny, M. Wagner, D. de Medici, A. Abdulmawjood, and P. Fach. 2003. Making internal amplification control mandatory for diagnostic PCR. J. Clin. Microbiol. 41:5835.
Hoorfar, J., B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach. 2004. Practical considerations in design of internal amplification controls for diagnostic PCR assays. J. Clin. Microbiol. 42:1863-1868.
Jantos, C. A., R. Roggendorf, F. N. Wuppermann, and J. H. Hegemann. 1998. Rapid detection of Chlamydia pneumoniae by PCR-enzyme immunoassay. J. Clin. Microbiol. 36:1890-1894.
Kolk, A. H. J., G. T. Noordhoek, O. De Leeuw, S. Kuijper, and J. D. A. Van Embden. 1994. Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantitation of bacteria. J. Clin. Microbiol. 32:1354-1356.
Lind-Brandberg, L., C. Welinder-Olsson, T. Lagergard, J. Taranger, B. Trollfors, and G. Zackrisson. 1998. Evaluation of PCR for diagnosis of Bordetella pertussis and Bordetella parapertussis infections. J. Clin. Microbiol. 36:679-683.
Martineau, F., F. J. Picard, P. H. Roy, M. Ouelette, and M. G. Bergeron. 1998. Species-specific and ubiquitous-DNA-based assays for rapid identification of Staphylococcus aureus. J. Clin. Microbiol. 36:618-623.
Mathis, A., and P. Deplazes. 1995. PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs. J. Clin. Microbiol. 33:1145-1149.
Morre, S. A., P. Sillekens, M. V. Jacobs, P. van Aarle, S. de Blok, B. van Gemen, J. M. Walboomers, C. J. L. M. Meijer, and A. J. van den Brule. 1996. RNA amplification by nucleic acid sequence-based amplification with an internal standard enables reliable detection of Chlamydia trachomatis in cervical scrapings and urine samples. J. Clin. Microbiol. 34:3108-3114.
Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.). 1999. Manual of clinical microbiology, 7th ed. ASM Press, Washington, D.C.
Nelson, S., A. Matlow, C. McDowell, M. Roscoe, M. Karmali, L. Penn, and L. Dyster. 1997. Detection of Bordetella pertussis in clinical specimens by PCR and microtiter plate-based DNA hybridization assay. J. Clin. Microbiol. 35:117-120.
Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim. 1985. Enzymatic amplification of -globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354.
Thoreson, A. C. E., M. Borre, L. P. Andersen, F. Jorgensen, S. Kiilerich, J. Scheidel, J. Rath, and K. A. Krogfelt. 1999. Helicobacter pylori detection in human biopsies: a competitive PCR assay with internal control reveals false results. FEMS Immunol. Med. Microbiol. 24:201-208.
Tjhie, T. J., F. J. van Kuppeveld, R. Roosendaal, W. J. Melchers, R. Gordijn, D. M. MacLaren, J. M. Walboomers, C. J. Meijer, and A. J. van den Brule. 1994. Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections. J. Clin. Microbiol. 32:11-16.
Ursi, J. P., D. Ursi, M. Ieven, and S. R. Pattyn. 1992. Utility of an internal control for the polymerase chain reaction. Application to detection of Mycoplasma pneumoniae in clinical specimens. APMIS 100:635-639.
Wadowsky, R. M., S. Laus, T. Libert, S. J. States, and G. D. Ehrlich. 1994. Inhibition of PCR-based assay for Bordetella pertussis by using calcium alginate fiber and aluminum shaft components of a nasopharyngeal swab. J. Clin. Microbiol. 32:1054-1057.
Whelen, A. C., and D. H. Persing. 1996. The role of nucleic acid amplification and detection in the clinical microbiology laboratory. Annu. Rev. Microbiol. 50:349-373.(Stephanie Herwegh, Christ)
INSERM E0364, Institut de Biologie de Lille, 1 rue du Pr. Calmette, 59021 Lille Cedex, France
Faculte des Sciences Pharmaceutiques et Biologiques, Lille, France
ABSTRACT
An internal control of amplification was constructed by recombinant PCR to detect PCR inhibitors. This exogenous DNA was included in the reaction mixture and coamplified with the target gene. This detection was successfully applied to the diagnosis of whooping cough by amplification of a fragment of Bordetella pertussis IS481.
TEXT
The detection of inhibitors of amplification remains a major problem which can partly be solved by the use of internal controls (ICs) of amplification (5, 6, 19). In addition to some commercially available systems, inhibitors can be detected either by spiking negative samples with the target DNA (7), by diluting the specimen to minimize the effects of inhibitors (16), or by adding a control template (2, 10, 15).
In this study, we developed for diagnosis of Bordetella pertussis a method to detect PCR inhibitors in clinical samples. We constructed an IC that can be amplified by the same primer pair as wild-type target DNA. IC was obtained by insertion of an exogenous DNA fragment into reference strain target by recombinant PCR (1, 3, 4).
B. pertussis Tohama phase I strain was the reference strain used to amplify the repetitive insertion sequence IS481. Escherichia coli DH5 was used as host for recombinant vector pMos (Amersham Pharmacia Biotech, Raiham, England). Amplified DNA from the B. pertussis reference strain was purified with a QIAquick PCR purification kit (QIAGEN, Hilden, Germany). This wild-type fragment was then directly used as template for recombinant PCR.
A 191-bp fragment from pertussis toxin gene amplified with primers Pt1 (5'-CCAACGCGCATGCGTGCAGATTGCTC-3') and Pt2 (5'-CCCTCTGCGTTTTGATGGTGCCTATTTTA-3') was introduced into the wild-type IS481 DNA (203 bp) by recombinant PCR (Fig. 1). Two internal primers, harboring a 5' end complementarity to the extremities of the exogenous toxin gene fragment to be inserted, were designed. In a first step, two PCR fragments were independently generated: fragment A with external primer A (Bp13, 5'-CCGCGCTGTGCCATGAGCTGG-3') and internal primer A (Pt1rv/Bp15, 5'-GACGAATCTGCACGCATGCGCGTTGGGGGCCCCGCAAGGCCGACTGGATGAAGCGTTCG-3') and fragment B with external primer B (Bp14, 5'-GATGCCTTGGTGGGGTCGATG-3') and internal primer B (Pt2rv/Bp16, 5'-TAAAATAGGCACCATCAAAACGCAGAGGGGGGCCCTGAGTGGGCTTACGCTCACACCTACCA-3'). These two amplicons and the 191-bp fragment were submitted to a second step of extension without primers. The third step consisted of a PCR with Bp13 and Bp14 in order to amplify the future internal control (Fig. 1). The resulting fragment (406 bp) was cloned into pMos vector. Recombinant plasmid was designated pSIBp. Construction was confirmed by PCR on thermolysates of E. coli strains containing the recombinant plasmid. Two ApaI sites flanking the exogenous DNA were present on Pt1rv/Bp15 and Pt2rv/Bp16 and were used to check the construction by ApaI restriction of the fragment amplified from an E. coli thermolysate. Three fragments of 100, 105, and 201 bp were obtained as expected. One clone of E. coli containing the correct recombinant plasmid was kept at –80°C. pSIBp plasmid was used as IC for detection in clinical samples.
IC concentration was optimized by adding various quantities of IC to PCRs containing constant amounts of wild-type DNA. Tenfold dilutions of the pSIBp plasmid were made, from 10 ng/μl to 10–10 ng/μl, and 5 μl was added to amplification reactions containing a constant amount of bacterial DNA known to be the threshold previously determined (103 CFU/ml). A 10–7 dilution of the pSIBp was determined as the optimal amount of IC. This dilution of plasmid corresponds to 104 molecules of internal standard versus 10 copies of the bacterial genome.
At high bacterial inoculums, only the wild-type fragment was amplified and no internal standard was detected (Fig. 2). Wild-type DNA is preferentially amplified because of its shorter length and because competition is minimized by addition of a small amount of IC. Since an excess of IC or genomic DNA may inhibit the amplification of the other by competition (Fig. 2), it is necessary to optimize the quantity of IC DNA added into the reaction mixtures. The amount of IC added in each reaction tube may seem quite high (104 plasmid copies) but is in accordance with previously published results (11).
Specimens for B. pertussis detection were nasopharyngeal aspirations (NPA) routinely received by the Bacteriology Laboratory of the Lille University Medical Center, France. The NPA collection tubing was flushed out by aspiration of 1 ml of sterile distilled water. Culture and identification of B. pertussis were performed according to conventional methods (13). For PCR detection, the NPA suspensions were boiled for 15 min and then centrifuged. Five microliters of the supernatant was directly used for amplification of IS481. Reaction mixtures were subjected to 35 cycles of amplification (30 s at 94°C, 30 s at 55°C, and 60 s at 72°C) (20). Amplicons were revealed by agarose gel electrophoresis followed by ethidium bromide staining.
One hundred twenty clinical samples were used to test the reliability of the B. pertussis IS481 IC. After a first amplification with pure NPA lysates, 33 samples (27.5%) were IS481 positive and 44 specimens (36.6%) were negative for IS481 but positive for the IC; in 43 samples (35.8%) neither the IC nor IS481 were detectable, suggesting the presence of inhibitors in the NPA samples. A second PCR assay was performed on these 43 NPA lysates, which were diluted 10-fold. Three specimens were IS481 positive, 25 specimens (20.8%) were negative for IS481 but positive for the IC, and 15 (12.5%) specimens remained inhibitory to the amplification of both IS481 and the IC. Following development tests, a 10-fold dilution of the thermolysate was included in our protocol for the diagnosis of B. pertussis.
A retrospective study over 4 years on 1,188 NPA specimens to detect B. pertussis by PCR was performed. The detection of IS481 was positive in 111 cases (9.3%), whereas 200 samples (16.8%) were negative for IS481 and the IC when tested with pure lysates but positive for the IC with 10-fold-diluted samples, 821 samples were negative for IS481 but positive for the IC, and 56 samples (4.7%) were negative for both IS481 and the IC. For the latter samples, PCRs obtained with DNA extracted with a commercial kit were still negative for IS481 but positive for IC. Although some authors (9, 14) have described no inhibition phenomenon within their clinical specimens, inhibition rates from 5 to 42% are quite usual (8, 16-18), depending on the nature of the clinical samples and the DNA extraction method. The present results, established on a large number of specimens, gave a smaller number of inhibiting specimens (21.5%) than the development study (35.8%) and were in agreement with those reported in the literature.
As the IC was amplified by the same primer pair used for the detection of the microorganism, there were no major modifications of the amplification protocols. Moreover, since the diagnosis of B. pertussis by IS481 was usually confirmed by a second PCR targeting pertussis toxin, our standardized IC, combining IS481 and pertussis toxin, was used as positive control for both assays. It should be possible to improve the amplification methods by addition of the IC into the clinical specimen prior to extraction (12). This IC could allow simultaneous detection of inefficient sample preparations.
ACKNOWLEDGMENTS
We are very grateful to A. Devalckenaere and S. Armand for their technical assistance.
REFERENCES
Donnenberg, M. S., J. Yu, and J. B. Kaper. 1993. A second chromosomal gene necessary for intimate attachment of enteropathogenic Escherichia coli to epithelial cells. J. Bacteriol. 175:4670-4680.
Gibb, A. P., and S. Wong. 1998. Inhibition of PCR by agar from bacteriological transport media. J. Clin. Microbiol. 36:275-276.
Higushi, R. 1990. Recombinant PCR, p. 177-183. In M. Innis (ed.), PCR protocols: a guide to methods and applications. Academic Press, Inc., San Diego, Calif.
Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59.
Hoorfar, J., N. Cook, B. Malorny, M. Wagner, D. de Medici, A. Abdulmawjood, and P. Fach. 2003. Making internal amplification control mandatory for diagnostic PCR. J. Clin. Microbiol. 41:5835.
Hoorfar, J., B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner, and P. Fach. 2004. Practical considerations in design of internal amplification controls for diagnostic PCR assays. J. Clin. Microbiol. 42:1863-1868.
Jantos, C. A., R. Roggendorf, F. N. Wuppermann, and J. H. Hegemann. 1998. Rapid detection of Chlamydia pneumoniae by PCR-enzyme immunoassay. J. Clin. Microbiol. 36:1890-1894.
Kolk, A. H. J., G. T. Noordhoek, O. De Leeuw, S. Kuijper, and J. D. A. Van Embden. 1994. Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantitation of bacteria. J. Clin. Microbiol. 32:1354-1356.
Lind-Brandberg, L., C. Welinder-Olsson, T. Lagergard, J. Taranger, B. Trollfors, and G. Zackrisson. 1998. Evaluation of PCR for diagnosis of Bordetella pertussis and Bordetella parapertussis infections. J. Clin. Microbiol. 36:679-683.
Martineau, F., F. J. Picard, P. H. Roy, M. Ouelette, and M. G. Bergeron. 1998. Species-specific and ubiquitous-DNA-based assays for rapid identification of Staphylococcus aureus. J. Clin. Microbiol. 36:618-623.
Mathis, A., and P. Deplazes. 1995. PCR and in vitro cultivation for detection of Leishmania spp. in diagnostic samples from humans and dogs. J. Clin. Microbiol. 33:1145-1149.
Morre, S. A., P. Sillekens, M. V. Jacobs, P. van Aarle, S. de Blok, B. van Gemen, J. M. Walboomers, C. J. L. M. Meijer, and A. J. van den Brule. 1996. RNA amplification by nucleic acid sequence-based amplification with an internal standard enables reliable detection of Chlamydia trachomatis in cervical scrapings and urine samples. J. Clin. Microbiol. 34:3108-3114.
Murray, P. R., E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.). 1999. Manual of clinical microbiology, 7th ed. ASM Press, Washington, D.C.
Nelson, S., A. Matlow, C. McDowell, M. Roscoe, M. Karmali, L. Penn, and L. Dyster. 1997. Detection of Bordetella pertussis in clinical specimens by PCR and microtiter plate-based DNA hybridization assay. J. Clin. Microbiol. 35:117-120.
Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim. 1985. Enzymatic amplification of -globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354.
Thoreson, A. C. E., M. Borre, L. P. Andersen, F. Jorgensen, S. Kiilerich, J. Scheidel, J. Rath, and K. A. Krogfelt. 1999. Helicobacter pylori detection in human biopsies: a competitive PCR assay with internal control reveals false results. FEMS Immunol. Med. Microbiol. 24:201-208.
Tjhie, T. J., F. J. van Kuppeveld, R. Roosendaal, W. J. Melchers, R. Gordijn, D. M. MacLaren, J. M. Walboomers, C. J. Meijer, and A. J. van den Brule. 1994. Direct PCR enables detection of Mycoplasma pneumoniae in patients with respiratory tract infections. J. Clin. Microbiol. 32:11-16.
Ursi, J. P., D. Ursi, M. Ieven, and S. R. Pattyn. 1992. Utility of an internal control for the polymerase chain reaction. Application to detection of Mycoplasma pneumoniae in clinical specimens. APMIS 100:635-639.
Wadowsky, R. M., S. Laus, T. Libert, S. J. States, and G. D. Ehrlich. 1994. Inhibition of PCR-based assay for Bordetella pertussis by using calcium alginate fiber and aluminum shaft components of a nasopharyngeal swab. J. Clin. Microbiol. 32:1054-1057.
Whelen, A. C., and D. H. Persing. 1996. The role of nucleic acid amplification and detection in the clinical microbiology laboratory. Annu. Rev. Microbiol. 50:349-373.(Stephanie Herwegh, Christ)