Development of a rapid recombinase polymerase amplification assay for the detection of Streptococcus pneumoniae in whole blood (original) (raw)
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Detection of Streptococcus pneumoniae in whole blood by PCR
Journal of clinical microbiology, 1995
Streptococcus pneumoniae is a major cause of bacteremia in both children and adults. Currently, the diagnosis of pneumococcal bacteremia relies on the isolation and identification of the bacteria from blood cultures. We have developed a sensitive assay for the detection of S. pneumoniae in whole blood by the PCR. A specific primer-probe set (JM201 and JM202 primers with JM204 probe) designed from the penicillin-binding protein 2B gene was demonstrated to reproducibly detect between 10 and 100 fg of input purified S. pneumoniae DNA. This assay system was shown to be inclusive for all strains of S. pneumoniae evaluated, including 15 different serotypes and a battery of penicillin-resistant and -sensitive strains. The specificity of this PCR-based assay was demonstrated by its inability to support amplification from a series of human, bacterial, and yeast genomic DNAs. A general specimen preparation method which should be suitable for the purification of DNA from any pathogens in whole...
Detection of Streptococcus pneumoniae DNA in blood cultures by PCR
Journal of clinical microbiology, 1994
We have developed a PCR assay, with primers derived from the autolysin (lyt) gene, for the detection of Streptococcus pneumoniae DNA in blood cultures. The predicted fragment of 247 bp was detected in all strains of pneumococci, embracing 12 different serotypes that were tested. Although DNA extracted from four viridans streptococci spp. Streptococcus oralis, Streptococcus mitis, Streptococcus sanguis, and Streptococcus parasanguis) gave amplification products, these were quite different from the predicted fragment for S. pneumoniae. Application of the assay for diagnosis of septicemia caused by S. pneumoniae showed concordance between PCR and culture results. However, on four occasions PCR was positive in supernatants from both paired culture bottles while pneumococci were cultured from only one. Performing PCR on negative cultures in controlled studies such as vaccine trials may provide a sensitive tool for increasing case detection.
Reports of biochemistry & molecular biology, 2020
Background Timely identification of Streptococcus pneumoniae infections can lead to a decrease in mortality rates. Differentiation of S. pneumoniae from other similar species using traditional culture-based and molecular methods is problematic. In this study, we assessed the efficacy of identifying the blpA and lytA for the detection of S. pneumoniae from isolates and various clinical samples using molecular methods. Methods A total of 440 clinical samples were collected from patients with suspected invasive pneumococcal infections during February 2016 to October 2018. Biochemical tests were used to confirm the dubious colonies on 5% sheep blood agar. Fifty-seven confirmed isolates, 57 culture-positive samples, and 57 culture-negative samples were analyzed for the presence of blpA and lytA using both conventional and real-time PCR. Results All the isolates and culture-positive samples were positive for blpA and lytA by both PCR methods. Of the 57 culture-negative samples, convention...
Journal of Clinical Microbiology, 2001
Molecular surveillance of pathogens has shown the need for rapid and dependable methods for the identification of organisms of clinical and epidemiological importance. As the leading cause of community-acquired pneumonia, Streptococcus pneumoniae was used as a model organism to develop and refine a real-time fluorescence PCR assay and enhanced DNA purification method. Seventy clinical isolates of S. pneumoniae, verified by latex agglutination, were screened against 26 negative control clinical isolates employing a TaqMan assay on a thermocycler (LightCycler). The probe, constructed from the lytA gene, correctly detected all S. pneumoniae genomes without cross-reaction to negative controls. The speed and ease of this approach will make it adaptable to identification of many bacterial pathogens and provide potential for adaptation to direct detection from patient specimens.
Development of PCRSeqTyping—a novel molecular assay for typing of Streptococcus pneumoniae
Pneumonia
Background: Precise serotyping of pneumococci is essential for vaccine development, to better understand the pathogenicity and trends of drug resistance. Currently used conventional and molecular methods of serotyping are expensive and time-consuming, with limited coverage of serotypes. An accurate and rapid serotyping method with complete coverage of serotypes is an urgent necessity. This study describes the development and application of a novel technology that addresses this need. Methods: Polymerase chain reaction (PCR) was performed, targeting 1061 bp cpsB region, and the amplicon was subjected to sequencing. The sequence data was analyzed using the National Centre for Biotechnology Information database. For homologous strains, a second round of PCR, sequencing, and data analysis was performed targeting 10 group-specific genes located in the capsular polysaccharide region. Ninety-one pneumococcal reference strains were analyzed with PCRSeqTyping and compared with Quellung reaction using Pneumotest Kit (SSI, Denmark).
Serotyping Streptococcus pneumoniae by Multiplex PCR
Journal of Clinical Microbiology, 2003
The capsule is a major virulence factor of pneumococci, and it was shown that some capsular variants are associated with antimicrobial resistance and certain types of disease. Moreover, pneumococcal capsular typing has received renewed interest since the availability of conjugate vaccines, which include serotypes frequently associated with pediatric disease. Our aim was to develop a simple, reliable, and economical method for detecting epidemiologically important serotypes present in the proposed 11-valent conjugate vaccine. We designed primers based on the sequences available for the capsular types 1, 3, 4, 6B, 14, 18C, 19F, 19A, and 23F and combined them into seven multiplex PCRs. The method involves streamlined DNA template preparation and agarose gel electrophoresis to analyze the amplification products. A total of 446 pneumococci selected from among isolates colonizing the nasopharynx of children attending day care centers in Lisbon, Portugal, were typed both by conventional immunological techniques and by multiplex PCR. Capsular types identified by the PCR method invariably produced results concordant with the conventional serotyping technique. Even when the method presented does not fully type an isolate, the PCR data can guide the experimenter when using immunological serotyping. Multiplex PCR for the analysis of pneumococci provides an accurate, expeditious, and cost-effective way of reducing the number of strains that have to be serotyped by conventional immunological techniques.
Streptococcus pneumoniae Serotyping by a Single Polymerase Chain Reaction–Based Multiplex Assay
Infectious Diseases in Clinical Practice, 2017
Background: Streptococcus pneumoniae is a prominent pathogen in children younger than 5 years as well as elderly people. Capsular serotyping of S. pneumoniae is necessary to develop the new vaccines and prevent invasive and noninvasive infections by S. pneumoniae. In this study, we used 2-step multiplex polymerase chain reaction (mPCR) that contained primers to detect PCV13 (13-valent pneumococcal conjugated vaccine) and non-PCV13 serotypes in different clinical and normal flora samples. Methods: A total of 100 S. pneumoniae isolates were obtained between 2013 and 2015 in Tehran, Iran. The sources of isolates were clinical and normal flora. Clinical isolates were eye infection (26%), blood (19%), sputum (18%), sinusitis and cerebrospinal fluid (9% each), trachea (7%), pleural aspirate (3%), otitis (3%), and urine, bronchoalveolar lavage, and abscess (2% each). Moreover, 43 normal flora isolates were collected from healthy individuals. The strain isolates were tested for antimicrobial susceptibility and serotyped by mPCR. Results: The highest rate of resistance was seen for trimethoprimsulfamethoxazole (96%) followed by tetracycline (77%), erythromycin (64%), clindamycin (56%), chloramphenicol (44%), and penicillin (26%). All isolates were susceptible to imipenem, ceftriaxone, vancomycin, linezolid, gemifloxacin, levofloxacin, moxifloxacin, and ofloxacin. By using mPCR, 91 and 7 isolates were typed in the first and second reactions, respectively. Two isolates were identified as nontypeable. The most frequent serotypes in 98 typeable serotypes were 23F (n = 21 [22%]), 14 (n = 19 [20%]), 3 (n = 13 [13%]), and 19F (n = 13 [13%]). Conclusions: Our multiplex assay is a precise and reliable method that can be used instead of the Quellung reaction for S. pneumoniae serotyping studies.
African Journal of Biotechnology, 2011
The aim of this study was to develop a real time polymerase chain reaction (PCR) for quantitative detection of Streptococcus pneumoniae from clinical respiratory specimens. Initially, 184 respiratory specimens from patients with community acquired pneumonia (CAP) (n = 129) and 55 cases with hospital associated pneumonia (HAP) were bacteriologically investigated. To check the colonization status among the healthy individuals, 32 preschool and 31 adults were screened in parallel. All specimens were cultured on selective culture media to isolate S. pneumoniae, Legionella spp. and Mycoplasma spp. A 166 bp fragment corresponding to cbp A gene of S. pneumoniae was amplified from clinical specimens using Taqman probe real time PCR. Culture showed 14, but real time PCR showed 15 specimens as being positive for S. pneumoniae. The specificity and sensitivity of real time PCR was 99.14% and 100 respectively. Co-infections of S. pneumoniae with Legionella pneumophila, Chlamydophila pneumoniae, Mycoplasma pneumoniae and Staphylococcus aureus were observed in 5 cases (35.72%). S. pneumoniae was counted <10 3 cfu/ml from the co-infected cases. Using real time PCR, a cutoff of 10 3 cfu/ml is introduced to differentiate colonization from infection in respiratory tract. This is the first report on the prevalence CAP with S. pneumoniae in Iran (12.40%).