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The detection of Bordetella pertussis in swab samples by polymerase chain reaction
The detection of Bordeteiia pertussis in swab samples by polymerase chain reaction F M Miillerl, Children’s *Division Bethesda,
Z M Li*, H J Schmitt’,
Hospital, of Bacterial MD, USA
Johannes Products,
M J Brennan*
Gutenberg-University, Center for Biologics
Mainz, Germany; Evaluation and Research,
FDA,
Summary Polymerase chain reaction (PCR) is revolutionizing the diagnosis of many infectious diseases, particularly those caused by organisms that are difficult to cultivate. Rapid and sensitive detection of Bordetella pertussis in clinical samples is important for the early diagnosis of pertussis, the prevention of transmission of the organisms and vaccine efficacy trials. In this study dacron and calcium-alginate nasopharyngeal swabs (NPSs) taken from patients with suspected pertussis were analysed by PCR using primers derived from the DNA adjacent to the 8. pertussis porin gene. NPSs in Regan-Lowe medium were transported to the laboratory for bacterial culture and PCR. Chelex 100 resin was used to extract bacterial DNA from NPSs. Three hundred and five NPSs including 127 dacron and 178 calcium-alginate swabs from 165 children with suspected pertussis were tested. Fourteen (11%) dacron swabs and 10 (6%) calcium-alginate swabs were culture positive. However, 23 (18%) dacron swabs and nine (5%) calcium-alginate swabs were positive by PCR using the ethidium bromide staining method. When the digoxigenin immunoblot system was used to increase the sensitivity of PCR, 79 (62%) dacron NPSs including 14 culture-positive samples were PCR positive; however, only 28 (16%) calcium-alginate NPSs including one culture-positive sample were positive by PCR. This data indicates that PCR is more sensitive than culture and the sensitivity of PCR can be increased using the digoxigenin immunoblot system. In addition, many fewer PCR positives were found in calcium-alginate swabs compared to dacron NPSs, which may be due to the quality and the Taq polymerase inhibitory effect of calcium-alginate fibre. Key words: immunoblot
Nasopharyngeal system
Serodiagn.
Immunother.
swabs,
Chelex
Infect. Disease
DNA extraction,
shared-primer
1994, Vol. 6, 230-232,
Introduction At present, bacterial culture is the routine technique used for the diagnosis of pertussis’4. However, the organism is slow-growing and in many clinical situations. such as the concurrent administration of antibiotics, isolation rates are low. Rapid, sensitive, and specific detection of Bordetella pertussis infection is important for effective therapy, prevention of the transmission of this organism, and vaccine efficacy
PCR, digoxigenin
December
triaW3. The polymerase chain reaction (PCR) is revolutionizing the diagnosis of many infectious diseases, particularly those caused by organisms that are difficult to cultivate2J. In this study, dacron and calcium-alginate nasopharyngeal swabs (NPSs) taken from patients with suspected pertussis were analysed by PCR. Materials
and methods
Bacterial culture Correspondence and reprint requests lo: Dr F-M Children’s Hospital, Pauwelsstrasse 30, D-52057 0 1994 Butterworth-Heinemann Ltd 088%0786/94/040230-03
Miiller, Aachen,
University Germany
Dacron and calcium-alginate nasopharyngeal swabs in Regan-Lowe transport medium containing Cephalexin (40 pg ml-‘) were sent to our laboratory by mail. Primary culture was done on Regan-Lowe agar plates
Mijller
et al.: Detection
of 6. pertussis
in swab
samples
by PCR
231
after NPSs had been pre-incubated at 37” C for 24 h. Culture was repeated after 48 h. The plates were examined for colonies after 48 h and then daily for 7 days. Suspicious colonies on the plates from primary or secondary bacterial culture were identified by colony morphology, Gram-staining and by slide agglutination using B. pevrussis-specific antiserum (Wellcome Diagnostics, Dartford, UK).
Table 1. Culture and PCR results nasopharyngeal swabs
Chelex 100 is a chelating resin that has a high affinity for polyvalent metal ions and was purchased from BioRad Laboratories (Munich. Germany). Chelex 100 is composed of styrene divinylbenzene copolymers containing paired iminodiacetate ions which act as chelating groups?.
and five NPSs including 127 dacron and 178 calciumalginate swabs were tested. PCR products were detected by ethidium bromide staining or by the digoxigenin immunoblot system. B. perrussis was cultured from 14 (11%) dacron and from 10 (6%) calciumalginate swabs. Twenty-three (18%) dacron NPSs and nine (5%) calcium-alginate NPSs were positive by PCR using the ethidium bromide staining method. When the digoxigenin immunoblot system was used, 79 (62%) dacron NPSs including all 14 culture-positive samples were PCR positive; however, only 28 (16%) calcium-alginate NPSs including one culture-positive sample gave a positive result (see Table 1).
Sample
preparfltion
Twenty per cent Chelex was used for bacterial DNA extraction. After pre-incubation at 37” C for 24 h, a swab containing nasopharyngeal secretion was placed in 300 ~1 phosphate buffered saline (PBS) and shaken vigorously for 5 min. The sample was then centrifuged at 14 000 rpm for 10 min in an Eppendorf centrifuge after NPS was removed. The pellet was resuspended in 200 ~1 20% Chelex 100 and boiled in a water bath for 10 min. The supernatant containing DNA was used for PCR after brief centrifugation. PCR PCR was performed essentially as described by Li et a1.j. A defined aliquot of the supernatant and digoxigenin-1 l-dUTP (Boehringer. Mannheim, Germany) were added into the PCR reaction mixture before DNA amplification. To prevent carryover contamination 0.5 U of uracil N-glycosylase (UNG) was added and 250 ,UM dUTP was substituted for 125 PM dTTP (UNG and dUTP were supplied in the carry-over prevention kit, Perkin Elmer, ijberlingen, Germany). Samples were incubated at 37” C for 10 min and then for 10 min at 94” C to inactivate the UNG and lyse the bacteria. Sharedprimer PCR was performed using primers derived from the DNA adjacent to the B. pertussis porin geneQ. Two-temperature DNA amplification was used for 40 cycles. Denaturation and annealing were carried out at 94” C and 60” C for 30 s. Positive controls of B. perrussis and B. pnrapertussis DNA and a negative control (dH,O) were included in each PCR run. A positive PCR result was detected by ethidium bromide staining of agarose gels after electrophoresis and by the digoxigenin
immunoblot
systemj.
Results
Dacron and calcium-alginate NPSs in Regan-Lowe transport medium were obtained from 165 children suspected of having pertussis infection. Three hundred
from
NPSs
No.
Culture
Ethidium bromide
Dacron Calciumalginate
127
14 (11%)
23 (18%)
178
10
(6%)
9
(5%)
305 immunoblot 79 (62%) 28 (16%)
Discussion
Procedures utilizing Chelex 100 resin have been developed for extracting DNA from forensic-type samples for use in PCR. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extractiorQ. Inhibitory factors from the transport medium are probably chelated by Chelex 100. The alkalinity of the Chelex 100 suspension (pH 10-11) and the exposure to 100” C temperature result in the lysis of the bacteria”. Schlgpfer et al. found the PCR to be inhibited in 26% of samples prepared without DNA extraction, whereas no inhibition was detected after DNA extraction’. The results of our study indicate that Chelex 100 is suitable for DNA extraction from dacron and calciumalginate NPSs in Regan-Lowe transport medium. The procedure is simple. rapid and effective and will help to reduce the chance of operator-introduced DNA contamination. PCR is a rapid. sensitive and specific technique for the detection of B. pertussis. Many different target genes and amplification primers are used in different studies+‘?. Li et al. developed a shared-primer PCR for the simultaneous detection of H. prrrtrssis and B. parapertussis from nasopharyngeal aspirates (NPAs). In that study the digoxigenin immunoblot system was used to increase the sensitivity of PCR. We used the same technique to detect B. pertussis in dacron and calcium-alginate swabs which were transported in Regan-Lowe medium. Twenty-three (18%) dacron NPSs and nine (5%) calcium-alginate NPSs were positive by PCR using the ethidium bromide staining
232
Serodiagn.
Immunother.
Infect.
Disease
1994; 6: No 4
method. When the digoxigenin immunoblot system was used, 79 (62%) dacron NPSs, including all 14 culture-positive samples, and 28 (16%) calciumalginate NPSs, including one culture-positive sample, were positive by PCR. These data indicate that PCR is more sensitive than culture and the sensitivity of PCR can be increased using the digoxigenin immunoblot system whether NPAs or NPSs are used. For isolation of B. pertussis by culture, calciumalginate swabs are superior to dacron, rayon, and cotton swabs’“. In contrast, in our study many fewer PCR positives were found in calcium-alginate swabs compared to dacron NPSs. This may be due to the quality of calcium-alginate swabs since 53 (30%) calcium-alginate NPSs were liquefied after they had been transported in Regan-Lowe medium. In addition, Wadowsky et al. have tested the effect of different types of swab on PCR-based detection of B. pertussis. They found that PCR was inhibited by using a calciumalginate fibre-tipped swab with an aluminium shaft but not by using a dacron fibre-tipped swab with a plastic shaftid. In conclusion we suggest that the dacron swab is superior to the calcium-alginate swab and should be used for collecting clinical samples for the detection of B. pertussis by PCR.
He Q, Mertsola J. Soini H, Viljanen MK. Sensitiveand specific polymerasechain reaction assaysfor detection of Bordetella pertussis in nasopharyngealspecimens.J Pediatrics 1994;124: 421-6 Li ZM, JansenDL, Finn TM, Halperin SA, Kasina A, O’Connor SP et al. Identification of Bordetella pertussis infection by shared-primerPCR. J Cfin Microbial
8
9
10 Part of these results have been presented at the 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, 1993, Abstr. 1562, p. 404; and the 94th ASM General Meeting, Las Vegas, 1994, Abstr. C-22, p. 494.
11
1994; 32: 783-9
Walsh PS, Metzger DA, Higuchi R. Chelex 100as a medium for simpleextraction of DNA for PCR-based typing from forensic material. BioTechniques 1991;10: 506-13 Li ZM. Hannah JH, Stibitz S. Nguyen NY, Manclark CR, Brennan MJ. Cloning and sequencingof the structural gene for the porin protein of Bordetella pertussis. Mel Microbial 1991;5: 1649-56 Schlapfer G, Senn HP, Berger M, Just M. Use of the polymerasechain reaction to detect Bordetella pertussis in patients with mild or atypical symptomsof infection. Eur J Clin Microbial Infect Dis 1993;12: lo-14 Douglas E. Coole JG, Parton R, McPheat W. Identification of Bordetella pertussis in nasopharyngeal swabsby PCR amplification of a region of the adenylate cyclasegene.J Med Microbiol 1993;38: 1404 Glare EM, Paton JC, Premier RR, Lawrence AJ, Nisbet IT. Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosisof pertussisusing the PolymeraseChain Reaction. J Clin Microbial 1990;28: 1982-7 Houard S, Hackel C. Herzog A, Bollen A. Specific identification of Bordetella pertussis by the Polymerase Chain Reaction. Res Microbial 1989;140: 477-87 Olcen P, Backman A, JohanssonB, Esbjorner E, Tornqvist E. Dygraves J, McPheat WL. Amplification of DNA by the PolymeraseChain Reaction for the efficient diagnosisof pertussis.Stand J Infect Dis 1992; 24: 339-45
References 1 Grimprel E, Begue P, Anjak I, Betsov F, Guiso N. Comparisonof polymerasechain reaction, culture and Western immunoblot serology for diagnosisof Bordetella pertussis infection. J Clin Microbial 1993;
12 van de Zee A, Agterbeerg C, PeetersM, Schellekens J, Rmooi F. Polymerasechain reaction assayfor pertussis:simultaneousdetection and discriminationof Bordetella pertussis and Bordetella parapertussis. J Clin Microbial 1993;31: 213440 13 Hoppe JE. WeissA. Recovery of Bordetella pertussis from four kinds of swabs.Eur J Clin Microbial 1987:
31: 2745.50 2 He Q, Mertsola J, Soini H, Skurnik M, Ruuskanen 0, Viljanen MK. Comparisonof polymerase chain reaction with culture and enzyme immunoassayfor diagnosisof pertussis.J Clin Microbial 1993;31: 642-5
6: 203-5 14
Wadowsky RM, Laus S, Libert T, States SJ, Ehrlich GD. Inhibition of PCR-basedassayfor Bordetella pertussis by usingcalcium alginate fiber and aluminium shaft componentsof a nasopharyngealswab.J Clin Microbiol 1994:32: 1054-7
Z M Li*, H J Schmitt’,
Hospital, of Bacterial MD, USA
Johannes Products,
M J Brennan*
Gutenberg-University, Center for Biologics
Mainz, Germany; Evaluation and Research,
FDA,
Summary Polymerase chain reaction (PCR) is revolutionizing the diagnosis of many infectious diseases, particularly those caused by organisms that are difficult to cultivate. Rapid and sensitive detection of Bordetella pertussis in clinical samples is important for the early diagnosis of pertussis, the prevention of transmission of the organisms and vaccine efficacy trials. In this study dacron and calcium-alginate nasopharyngeal swabs (NPSs) taken from patients with suspected pertussis were analysed by PCR using primers derived from the DNA adjacent to the 8. pertussis porin gene. NPSs in Regan-Lowe medium were transported to the laboratory for bacterial culture and PCR. Chelex 100 resin was used to extract bacterial DNA from NPSs. Three hundred and five NPSs including 127 dacron and 178 calcium-alginate swabs from 165 children with suspected pertussis were tested. Fourteen (11%) dacron swabs and 10 (6%) calcium-alginate swabs were culture positive. However, 23 (18%) dacron swabs and nine (5%) calcium-alginate swabs were positive by PCR using the ethidium bromide staining method. When the digoxigenin immunoblot system was used to increase the sensitivity of PCR, 79 (62%) dacron NPSs including 14 culture-positive samples were PCR positive; however, only 28 (16%) calcium-alginate NPSs including one culture-positive sample were positive by PCR. This data indicates that PCR is more sensitive than culture and the sensitivity of PCR can be increased using the digoxigenin immunoblot system. In addition, many fewer PCR positives were found in calcium-alginate swabs compared to dacron NPSs, which may be due to the quality and the Taq polymerase inhibitory effect of calcium-alginate fibre. Key words: immunoblot
Nasopharyngeal system
Serodiagn.
Immunother.
swabs,
Chelex
Infect. Disease
DNA extraction,
shared-primer
1994, Vol. 6, 230-232,
Introduction At present, bacterial culture is the routine technique used for the diagnosis of pertussis’4. However, the organism is slow-growing and in many clinical situations. such as the concurrent administration of antibiotics, isolation rates are low. Rapid, sensitive, and specific detection of Bordetella pertussis infection is important for effective therapy, prevention of the transmission of this organism, and vaccine efficacy
PCR, digoxigenin
December
triaW3. The polymerase chain reaction (PCR) is revolutionizing the diagnosis of many infectious diseases, particularly those caused by organisms that are difficult to cultivate2J. In this study, dacron and calcium-alginate nasopharyngeal swabs (NPSs) taken from patients with suspected pertussis were analysed by PCR. Materials
and methods
Bacterial culture Correspondence and reprint requests lo: Dr F-M Children’s Hospital, Pauwelsstrasse 30, D-52057 0 1994 Butterworth-Heinemann Ltd 088%0786/94/040230-03
Miiller, Aachen,
University Germany
Dacron and calcium-alginate nasopharyngeal swabs in Regan-Lowe transport medium containing Cephalexin (40 pg ml-‘) were sent to our laboratory by mail. Primary culture was done on Regan-Lowe agar plates
Mijller
et al.: Detection
of 6. pertussis
in swab
samples
by PCR
231
after NPSs had been pre-incubated at 37” C for 24 h. Culture was repeated after 48 h. The plates were examined for colonies after 48 h and then daily for 7 days. Suspicious colonies on the plates from primary or secondary bacterial culture were identified by colony morphology, Gram-staining and by slide agglutination using B. pevrussis-specific antiserum (Wellcome Diagnostics, Dartford, UK).
Table 1. Culture and PCR results nasopharyngeal swabs
Chelex 100 is a chelating resin that has a high affinity for polyvalent metal ions and was purchased from BioRad Laboratories (Munich. Germany). Chelex 100 is composed of styrene divinylbenzene copolymers containing paired iminodiacetate ions which act as chelating groups?.
and five NPSs including 127 dacron and 178 calciumalginate swabs were tested. PCR products were detected by ethidium bromide staining or by the digoxigenin immunoblot system. B. perrussis was cultured from 14 (11%) dacron and from 10 (6%) calciumalginate swabs. Twenty-three (18%) dacron NPSs and nine (5%) calcium-alginate NPSs were positive by PCR using the ethidium bromide staining method. When the digoxigenin immunoblot system was used, 79 (62%) dacron NPSs including all 14 culture-positive samples were PCR positive; however, only 28 (16%) calcium-alginate NPSs including one culture-positive sample gave a positive result (see Table 1).
Sample
preparfltion
Twenty per cent Chelex was used for bacterial DNA extraction. After pre-incubation at 37” C for 24 h, a swab containing nasopharyngeal secretion was placed in 300 ~1 phosphate buffered saline (PBS) and shaken vigorously for 5 min. The sample was then centrifuged at 14 000 rpm for 10 min in an Eppendorf centrifuge after NPS was removed. The pellet was resuspended in 200 ~1 20% Chelex 100 and boiled in a water bath for 10 min. The supernatant containing DNA was used for PCR after brief centrifugation. PCR PCR was performed essentially as described by Li et a1.j. A defined aliquot of the supernatant and digoxigenin-1 l-dUTP (Boehringer. Mannheim, Germany) were added into the PCR reaction mixture before DNA amplification. To prevent carryover contamination 0.5 U of uracil N-glycosylase (UNG) was added and 250 ,UM dUTP was substituted for 125 PM dTTP (UNG and dUTP were supplied in the carry-over prevention kit, Perkin Elmer, ijberlingen, Germany). Samples were incubated at 37” C for 10 min and then for 10 min at 94” C to inactivate the UNG and lyse the bacteria. Sharedprimer PCR was performed using primers derived from the DNA adjacent to the B. pertussis porin geneQ. Two-temperature DNA amplification was used for 40 cycles. Denaturation and annealing were carried out at 94” C and 60” C for 30 s. Positive controls of B. perrussis and B. pnrapertussis DNA and a negative control (dH,O) were included in each PCR run. A positive PCR result was detected by ethidium bromide staining of agarose gels after electrophoresis and by the digoxigenin
immunoblot
systemj.
Results
Dacron and calcium-alginate NPSs in Regan-Lowe transport medium were obtained from 165 children suspected of having pertussis infection. Three hundred
from
NPSs
No.
Culture
Ethidium bromide
Dacron Calciumalginate
127
14 (11%)
23 (18%)
178
10
(6%)
9
(5%)
305 immunoblot 79 (62%) 28 (16%)
Discussion
Procedures utilizing Chelex 100 resin have been developed for extracting DNA from forensic-type samples for use in PCR. The extraction of DNA from semen and very small bloodstains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extractiorQ. Inhibitory factors from the transport medium are probably chelated by Chelex 100. The alkalinity of the Chelex 100 suspension (pH 10-11) and the exposure to 100” C temperature result in the lysis of the bacteria”. Schlgpfer et al. found the PCR to be inhibited in 26% of samples prepared without DNA extraction, whereas no inhibition was detected after DNA extraction’. The results of our study indicate that Chelex 100 is suitable for DNA extraction from dacron and calciumalginate NPSs in Regan-Lowe transport medium. The procedure is simple. rapid and effective and will help to reduce the chance of operator-introduced DNA contamination. PCR is a rapid. sensitive and specific technique for the detection of B. pertussis. Many different target genes and amplification primers are used in different studies+‘?. Li et al. developed a shared-primer PCR for the simultaneous detection of H. prrrtrssis and B. parapertussis from nasopharyngeal aspirates (NPAs). In that study the digoxigenin immunoblot system was used to increase the sensitivity of PCR. We used the same technique to detect B. pertussis in dacron and calcium-alginate swabs which were transported in Regan-Lowe medium. Twenty-three (18%) dacron NPSs and nine (5%) calcium-alginate NPSs were positive by PCR using the ethidium bromide staining
232
Serodiagn.
Immunother.
Infect.
Disease
1994; 6: No 4
method. When the digoxigenin immunoblot system was used, 79 (62%) dacron NPSs, including all 14 culture-positive samples, and 28 (16%) calciumalginate NPSs, including one culture-positive sample, were positive by PCR. These data indicate that PCR is more sensitive than culture and the sensitivity of PCR can be increased using the digoxigenin immunoblot system whether NPAs or NPSs are used. For isolation of B. pertussis by culture, calciumalginate swabs are superior to dacron, rayon, and cotton swabs’“. In contrast, in our study many fewer PCR positives were found in calcium-alginate swabs compared to dacron NPSs. This may be due to the quality of calcium-alginate swabs since 53 (30%) calcium-alginate NPSs were liquefied after they had been transported in Regan-Lowe medium. In addition, Wadowsky et al. have tested the effect of different types of swab on PCR-based detection of B. pertussis. They found that PCR was inhibited by using a calciumalginate fibre-tipped swab with an aluminium shaft but not by using a dacron fibre-tipped swab with a plastic shaftid. In conclusion we suggest that the dacron swab is superior to the calcium-alginate swab and should be used for collecting clinical samples for the detection of B. pertussis by PCR.
He Q, Mertsola J. Soini H, Viljanen MK. Sensitiveand specific polymerasechain reaction assaysfor detection of Bordetella pertussis in nasopharyngealspecimens.J Pediatrics 1994;124: 421-6 Li ZM, JansenDL, Finn TM, Halperin SA, Kasina A, O’Connor SP et al. Identification of Bordetella pertussis infection by shared-primerPCR. J Cfin Microbial
8
9
10 Part of these results have been presented at the 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, 1993, Abstr. 1562, p. 404; and the 94th ASM General Meeting, Las Vegas, 1994, Abstr. C-22, p. 494.
11
1994; 32: 783-9
Walsh PS, Metzger DA, Higuchi R. Chelex 100as a medium for simpleextraction of DNA for PCR-based typing from forensic material. BioTechniques 1991;10: 506-13 Li ZM. Hannah JH, Stibitz S. Nguyen NY, Manclark CR, Brennan MJ. Cloning and sequencingof the structural gene for the porin protein of Bordetella pertussis. Mel Microbial 1991;5: 1649-56 Schlapfer G, Senn HP, Berger M, Just M. Use of the polymerasechain reaction to detect Bordetella pertussis in patients with mild or atypical symptomsof infection. Eur J Clin Microbial Infect Dis 1993;12: lo-14 Douglas E. Coole JG, Parton R, McPheat W. Identification of Bordetella pertussis in nasopharyngeal swabsby PCR amplification of a region of the adenylate cyclasegene.J Med Microbiol 1993;38: 1404 Glare EM, Paton JC, Premier RR, Lawrence AJ, Nisbet IT. Analysis of a repetitive DNA sequence from Bordetella pertussis and its application to the diagnosisof pertussisusing the PolymeraseChain Reaction. J Clin Microbial 1990;28: 1982-7 Houard S, Hackel C. Herzog A, Bollen A. Specific identification of Bordetella pertussis by the Polymerase Chain Reaction. Res Microbial 1989;140: 477-87 Olcen P, Backman A, JohanssonB, Esbjorner E, Tornqvist E. Dygraves J, McPheat WL. Amplification of DNA by the PolymeraseChain Reaction for the efficient diagnosisof pertussis.Stand J Infect Dis 1992; 24: 339-45
References 1 Grimprel E, Begue P, Anjak I, Betsov F, Guiso N. Comparisonof polymerasechain reaction, culture and Western immunoblot serology for diagnosisof Bordetella pertussis infection. J Clin Microbial 1993;
12 van de Zee A, Agterbeerg C, PeetersM, Schellekens J, Rmooi F. Polymerasechain reaction assayfor pertussis:simultaneousdetection and discriminationof Bordetella pertussis and Bordetella parapertussis. J Clin Microbial 1993;31: 213440 13 Hoppe JE. WeissA. Recovery of Bordetella pertussis from four kinds of swabs.Eur J Clin Microbial 1987:
31: 2745.50 2 He Q, Mertsola J, Soini H, Skurnik M, Ruuskanen 0, Viljanen MK. Comparisonof polymerase chain reaction with culture and enzyme immunoassayfor diagnosisof pertussis.J Clin Microbial 1993;31: 642-5
6: 203-5 14
Wadowsky RM, Laus S, Libert T, States SJ, Ehrlich GD. Inhibition of PCR-basedassayfor Bordetella pertussis by usingcalcium alginate fiber and aluminium shaft componentsof a nasopharyngealswab.J Clin Microbiol 1994:32: 1054-7