Modified multiplex PCR method for detection of pyrogenic exotoxin genes in staphylococcal isolates.
Ontology highlight
ABSTRACT:
SUBMITTER: Lovseth A
PROVIDER: S-EPMC497657 | biostudies-literature | 2004 Aug
REPOSITORIES: biostudies-literature
Publications
Modified multiplex PCR method for detection of pyrogenic exotoxin genes in staphylococcal isolates.
Løvseth Astrid A Loncarevic Semir S Berdal Knut G KG
Journal of clinical microbiology 20040801 8
A modified multiplex PCR method for detection of nine Staphylococcus aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and one form of immunoreactive toxic shock syndrome toxin based on a previously published method (S. R. Monday and G. A. Bohach, J. Clin. Microbiol. 37:3411-3414, 1999) has been developed. The modified PCR protocol seems robust and gives reliable results. ...[more]
Similar Datasets
Project description:Staphylococcus aureus may contain one or more genes that encode a variety of immunomodulatory pyrogenic toxins (PTs), including the staphylococcal enterotoxins and toxic shock syndrome toxin (TSST). The PTs interact with several cellular targets to produce disease, such as food poisoning and toxic shock syndrome. At present, nine serologically distinct enterotoxins and one immunoreactive form of TSST have been identified and characterized. As isolates of S. aureus are further assessed, it is anticipated that this number will increase. To facilitate screening, a multiplex PCR was designed to simultaneously determine which of these 10 currently known PT genes an individual S. aureus isolate possesses. We show here, using S. aureus isolates with characterized PT phenotypes, that this novel PCR technique reliably detects each of the known PTs in a single reaction.
| S-EPMC85588 | biostudies-literature
Project description:A novel multiplex PCR method using three sets of specific primers was developed for the detection of the cytotoxic (act), heat-labile (alt), and heat-stable (ast) enterotoxin genes in Aeromonas spp. This assay was used to characterize 35 reference strains as well as 537 food-borne isolates. A total of seven gene pattern combinations were encountered, including act, alt, act/alt, act/alt/ast, act/alt/148-bp amplicon, alt/ast, and alt/148-bp amplicon. The alt gene was detected with 34 reference strains (97%) and occurred singly in 14% of these strains. The frequency of occurrence of the act/alt, act/alt/ast, and alt/ast gene patterns in reference strains was 14 (40%), 2 (6%), and 2 (6%), respectively. An unpredicted amplicon was detected in 11 reference strains (31%). Characterization of this amplicon showed that its size was 148 bp, as generated by the AHLF and AHLR primers, and that it uniquely aligned with the Aeromonas salmonicida A449 genome sequence (GenBank accession number CP000644). This amplicon was named Aeromonas salmonicida subsp. salmonicida hypothetical protein amplicon (AssHPA). In the 537 food-borne isolates, the act and alt genes were most dominant and were detected in 349 (65%) and 452 (84%) isolates, respectively, either alone or in combinations. The act and alt genes occurred singly in 30 (6%) and 128 (24%) of these strains, respectively. The act/alt gene pattern occurred in 315 isolates (59%), whereas the ast gene was always linked to strains exhibiting the act/alt/ast and alt/ast gene combinations in 4 (0.7%) and 5 (0.9%) isolates, respectively. The uniplex amplification of three enterotoxin genes separately confirms the specificity of the unique selected primers. This multiplex PCR is rapid and simple and can detect the presence of three Aeromonas enterotoxin genes in a single assay.
| S-EPMC2805215 | biostudies-literature
Project description:Therapeutic options for infections caused by gram-negative organisms expressing plasmid-mediated AmpC beta-lactamases are limited because these organisms are usually resistant to all the beta-lactam antibiotics, except for cefepime, cefpirome, and the carbapenems. These organisms are a major concern in nosocomial infections and should therefore be monitored in surveillance studies. Six families of plasmid-mediated AmpC beta-lactamases have been identified, but no phenotypic test can differentiate among them, a fact which creates problems for surveillance and epidemiology studies. This report describes the development of a multiplex PCR for the purpose of identifying family-specific AmpC beta-lactamase genes within gram-negative pathogens. The PCR uses six sets of ampC-specific primers resulting in amplicons that range from 190 bp to 520 bp and that are easily distinguished by gel electrophoresis. ampC multiplex PCR differentiated the six plasmid-mediated ampC-specific families in organisms such as Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, and Salmonella enterica serovar Typhimurium. Family-specific primers did not amplify genes from the other families of ampC genes. Furthermore, this PCR-based assay differentiated multiple genes within one reaction. In addition, WAVE technology, a high-pressure liquid chromatography-based separation system, was used as a way of decreasing analysis time and increasing the sensitivity of multiple-gene assays. In conclusion, a multiplex PCR technique was developed for identifying family-specific ampC genes responsible for AmpC beta-lactamase expression in organisms with or without a chromosomal AmpC beta-lactamase gene.
| S-EPMC130804 | biostudies-literature
Project description:We report the development of a multiplex PCR protocol for the diagnosis of staphylococcal infection. The protocol was designed to (i) detect any staphylococcal species to the exclusion of other bacterial pathogens (based on primers corresponding to Staphylococcus-specific regions of the 16S rRNA genes), (ii) distinguish between S. aureus and the coagulase-negative staphylococci (CNS) (based on amplification of the S. aureus-specific clfA gene), and (iii) provide an indication of the likelihood that the staphylococci present in the specimen are resistant to oxacillin (based on amplification of the mecA gene). The expected fragments were amplified from each of 60 staphylococcal isolates (13 oxacillin-resistant S. aureus isolates, 23 oxacillin-sensitive S. aureus isolates, 17 oxacillin-resistant CNS, and 7 oxacillin-sensitive CNS). No amplification products were observed with template DNA from nonstaphylococcal species, and the efficiency of amplification of staphylococcal targets was not adversely affected by the presence of DNA from other bacterial species in the same sample. The utility of the protocol for the analysis of clinical samples was verified by analysis of aliquots taken directly from BacT/Alert blood culture bottles. Of 77 blood cultures tested, only 7 yielded results inconsistent with those of conventional methods of diagnosis and susceptibility testing. Of those, one was identified as a CNS species by PCR and S. aureus by conventional methods. We also identified two isolates that were mecA positive but were oxacillin sensitive according to conventional methods. The other four samples failed to yield any amplification product even with a control set of primers corresponding to a conserved region of the eubacterial rRNA genes.
| S-EPMC88340 | biostudies-literature
Project description:Endoribonuclease Y (RNase Y) is a crucial regulator of virulence in Gram-positive bacteria. In the human pathogen Streptococcus pyogenes, RNase Y is required for the expression of the major secreted virulence factor streptococcal pyrogenic exotoxin B (SpeB), but the mechanism involved in this regulation remains elusive. Here, we demonstrate that the 5' untranslated region of speB mRNA is processed by several RNases including RNase Y. In particular, we identify two RNase Y cleavage sites located downstream of a guanosine (G) residue. To assess whether this nucleotide is required for RNase Y activity in vivo, we mutated it and demonstrate that the presence of this G residue is essential for the processing of the speB mRNA 5' UTR by RNase Y. Although RNase Y directly targets and processes speB, we show that RNase Y-mediated regulation of speB expression occurs primarily at the transcriptional level and independently of the processing in the speB mRNA 5' UTR. To conclude, we demonstrate for the first time that RNase Y processing of an mRNA target requires the presence of a G. We also provide new insights on the speB 5' UTR and on the role of RNase Y in speB regulation.
| S-EPMC6284565 | biostudies-literature
Project description:Streptococcal toxic shock syndrome (STSS) is a highly lethal, acute-onset illness that is a subset of invasive streptococcal disease. The majority of clinical STSS cases have been associated with the pyrogenic toxin superantigens (PTSAgs) streptococcal pyrogenic exotoxin A or C (SPE A or C), although cases have been reported that are not associated with either of these exotoxins. Recent genome sequencing projects have revealed a number of open reading frames that potentially encode proteins with similarity to SPEs A and C and to other PTSAgs. Here, we describe the cloning, expression, purification, and functional characterization of a novel exotoxin termed streptococcal pyrogenic exotoxin J (SPE J). Purified recombinant SPE J (rSPE J) expressed from Escherichia coli stimulated the expansion of both rabbit splenocytes and human peripheral blood lymphocytes, preferentially expanded human T cells displaying Vbeta2, -3, -12, -14, and -17 on their T-cell receptors, and was active at concentrations as low as 5 x 10(-6) microg/ml. Furthermore, rSPE J induced fevers in rabbits and was lethal in two models of STSS. Biochemically, SPE J had a predicted molecular weight of 24,444 and an isoelectric point of 7.7 and lacked the ability to form the cystine loop structure characteristic of many PTSAgs. SPE J shared 19.6, 47.1, 38.8, 18.1, 19.6, and 24.4% identity with SPEs A, C, G, and H, streptococcal superantigen, and streptococcal mitogenic exotoxin Z-2, respectively, and was immunologically cross-reactive with SPE C. The characterization of a seventh functional streptococcal PTSAg raises important questions relating to the evolution of the streptococcal superantigens.
| S-EPMC98031 | biostudies-literature
Project description:BACKGROUND:Vancomycin Resistant Enterococci (VRE) are a major cause of nosocomial infections. There are various phenotypic and genotypic methods of detection of glycopeptide resistance in enterococci. This study utilizes multiplex PCR for reliable detection of various glycopeptides resistance genes in VRE. METHOD:This study was conducted to detect and to assess the prevalence of vancomycin resistance among enterococci isolates. From October 2011 to June 2013, a total of 96 non-repetitive isolates of enterococci from various clinical samples were analyzed. VRE were identified by Kirby Bauer disc diffusion method with Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) of all isolates for vancomycin and teicoplanin was determined by E-test. Multiplex PCR was carried out for all enterococci isolates using six sets of primers. RESULTS:Out of 96 isolates, 14 (14.6%) were found to be resistant to vancomycin by vancomycin E-test method (MIC ?32 ?g/ml). Out of these 14 isolates, 13 were also resistant to teicoplanin (MIC ?16 ?g/ml). VanA gene was detected in all the 14 isolates by Multiplex PCR. One of the PCR amplicons was sent for sequencing and the sequence received was submitted in the GenBank (GenBank accession no. KF181100). CONCLUSION:Prevalence of VRE in this study was 14.6%. Multiplex PCR is a robust, sensitive and specific technique, which can be used for rapid detection of various glycopeptide resistance genes. Rapid identification of patients infected or colonized with VRE is essential for implementation of appropriate control measures to prevent their spread.
| S-EPMC4297841 | biostudies-literature
Project description:Rapid and accurate identification of patients colonised with carbapenemase-producing organisms (CPOs) is essential to adopt prompt prevention measures to reduce the risk of transmission. Recent studies have demonstrated the ability to combine machine learning (ML) algorithms with real-time digital PCR (dPCR) instruments to increase classification accuracy of multiplex PCR assays when using synthetic DNA templates. We sought to determine if this novel methodology could be applied to improve identification of the five major carbapenem-resistant genes in clinical CPO-isolates, which would represent a leap forward in the use of PCR-based data-driven diagnostics for clinical applications. We collected 253 clinical isolates (including 221 CPO-positive samples) and developed a novel 5-plex PCR assay for detection of blaIMP, blaKPC, blaNDM, blaOXA-48, and blaVIM. Combining the recently reported ML method "Amplification and Melting Curve Analysis" (AMCA) with the abovementioned multiplex assay, we assessed the performance of the AMCA methodology in detecting these genes. The improved classification accuracy of AMCA relies on the usage of real-time data from a single-fluorescent channel and benefits from the kinetic/thermodynamic information encoded in the thousands of amplification events produced by high throughput real-time dPCR. The 5-plex showed a lower limit of detection of 10 DNA copies per reaction for each primer set and no cross-reactivity with other carbapenemase genes. The AMCA classifier demonstrated excellent predictive performance with 99.6% (CI 97.8-99.9%) accuracy (only one misclassified sample out of the 253, with a total of 160,041 positive amplification events), which represents a 7.9% increase (p-value <0.05) compared to conventional melting curve analysis. This work demonstrates the use of the AMCA method to increase the throughput and performance of state-of-the-art molecular diagnostic platforms, without hardware modifications and additional costs, thus potentially providing substantial clinical utility on screening patients for CPO carriage.
| S-EPMC8650054 | biostudies-literature
Project description:In this study, we describe a multiplex PCR to detect a AGC-->ACC (serine to threonine) mutation in the katG gene and a -15 C-to-T substitution (inhA(C-15T)) at the 5' end of a presumed ribosome binding site in the promoter of the mabA-inhA operon. These mutations have been reported in the majority of previous studies as the most frequent mutations involved in the resistance to isoniazid (INH) of Mycobacterium tuberculosis clinical strains with high levels of resistance. The method was optimized and validated after an analysis of 30 M. tuberculosis clinical isolates with known sequences of the relevant part of the katG gene and the regulatory region of the mabA-inhA operon. We analyzed 297 INH-resistant M. tuberculosis isolates collected in Spain from 1996 to 2003 by PCR-restriction fragment length polymorphism (using the katG gene), DNA sequencing, and the newly developed multiplex PCR. The results were concordant for all 297 isolates tested. The analysis revealed that 204 (68.7%) of the isolates carried one or both of the mutations. This finding suggests that with further development this multiplex PCR will be able to detect the majority of the INH-resistant M. tuberculosis clinical isolates from Spain and other countries where a high frequency of similar mutations occur.
| S-EPMC538849 | biostudies-literature
Project description:WGS Staphylococcal isolates from PJI and Wounds
| PRJNA765573 | ENA