Detection of Bordetella pertussis in clinical specimens by PCR and a microtiter plate-based DNA hybridization assay

Abstract

In order to improve detection of Bordetella pertussis in nasopharyngeal aspirates (NPAs) in our laboratory, a PCR-based assay was optimized, and a study was designed (i) to compare results obtained by PCR to those obtained by culture and (ii) to evaluate a novel microtiter plate-based DNA hybridization assay (PCR-plate) by comparing it to agarose gel electrophoresis (PCR-gel) for detection of the PCR product. DNA for the PCR was extracted with a guanidine thiocyanate buffer and used in a PCR mixture containing primers directed against a reiterated gene sequence in B. pertussis (Q. He, J. Mertsola, H. Soini, M. Skurnik, O. Ruuskanen, and M. K. Viljanen, J. Clin. Microbiol. 31:642-645, 1993). Of 96 NPAs submitted from a targeted study group, 23 were positive by culture, 27 were positive by PCR-gel, and 31 were positive by PCR-plate. All culture-positive specimens were also positive by PCR. Of nine patients with culture-negative-PCR-positive results, six had discharge diagnoses of pertussis. Thus, PCR with plate-based product detection is a sensitive method for the laboratory detection of B. pertussis in NPAs. Additional advantages of the plate assay include rapidity, objectivity in reading results, specificity, and the capability of being adapted to a high-volume, automated system.

Comparison of PCR, Culture, and Direct Fluorescent-Antibody Testing for Detection of Bordetella pertussis

“…(i) DNA extraction. DNA was extracted from NP swabs by a modification of the procedure described by Nelson et al (21). Four hundred microliters of lysis reagent (5 M guanidine thiocyanate; 50 mM Tris [pH 7.6]; 50 g of glycogen from mussels [Sigma Chemical Co.] per ml, and 100 mM dithiothreitol) was added to microcentrifuge tubes containing NP swabs.…”

Comprehensive PCR-Based Assay for Detection and Species Identification of Human Herpesviruses

“…(i) CSF. DNA was extracted from cerebrospinal fluid (CSF) by a guanidine thiocyanate (GTC) method (23), as follows. To 100 l of CSF were added 400 l of extraction buffer (5.75 M guanidine thiocyanate [G9277; Sigma], 50 mM Tris [pH 7.4], 50 g of glycogen [901393; Boehringer-Mannheim] per ml, 10 l of ␤-mercaptoethanol [Sigma] per ml).…”

Detection of Ureaplasma urealyticum in Endotracheal Tube Aspirates from Neonates by PCR

“…The PCR inhibition rate, as assessed by the lack of amplification of a low copy number of U. urealyticum DNA targets added to negative specimens, was reasonably low in this study, at less than 7% of specimens. We have previously reported the utility of the guanidine isothiocyanate-based DNA extraction procedure for use with respiratory specimens (8). In specimen extracts that contain inhibitors, inhibition may be overcome by a 1-in-5 or a 1-in-10 dilution of the DNA in water in the majority of cases.…”

“…A guanidine isothiocyanate extraction procedure with isopropanol precipitation was used to obtain DNA from the specimens. We have previously demonstrated the utility of this procedure with respiratory specimens (8), and it is the basis for many commercialized DNA extraction kits. To 100-l specimen aliquots, 4 volumes of extraction buffer was added (5.75 M guanidine thiocyanate, 50 mM Tris [pH 7.4], 50 g of glycogen per ml, and 1% ␤-mercaptoethanol [added immediately prior to use]), and the mixture was vortexed and incubated at room temperature for 10 min.…”