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Survey of nasal colonization by, and assessment of a novel multiplex PCR method for detection of biofilm-forming methicillin-resistant staphylococci in healthy medical students.

We surveyed the prevalence of nasal colonization by biofilm-forming methicillin-resistant staphylococci in healthy medical students, who had never had contact with patients, using polymerase chain reaction (PCR) to detect themec A gene, production of penicillin-binding protein 2' (PBP2'), and quantitative assay of biofilm formation on polystyrene. Anterior nasal swabs from 90 students were cultured on mannitol salt and oxacillin salt screening agar plates. In total, 231 staphylococcal isolates belonging to 10 species from 88 students were identified, of which 139 from 77 (88%) students were Staphylococcus epidermidis. The overall prevalences of methicillin-resistant and biofilm-forming staphylococci were 48% (43 of 90) and 59% (53 of 90) for the medical students, respectively. In total 30 (33%) students carried biofilm-forming methicillin-resistant staphylococci in the nares, all of which were identified as S. epidermidis. For rapid detection of biofilm-forming methicillin-resistant S. epidermidis (MRSE), we devised a novel multiplex PCR method to assess a total of 243 staphylococcal isolates, including the 231 isolates from the students. The multiplex PCR assay used six primers to amplify atl E and ica ADB, which are responsible for the biofilm formation ofS. epidermidis, and mec A genes. The multiplex PCR assay revealed that 68 (96%) isolates were detectable in 71 biofilm-forming MRSE isolates, which corresponded to 93% (28 of 30) of biofilm-forming MRSE carriers. Surveillance of nasal colonization with biofilm-forming MRSE using this multiplex PCR in healthcare workers and patients, might provide useful information for the establishment of infection control procedures toward biofilm-forming MRSE.

Adult↗

Multiplex PCR of bcr-abl fusion transcripts in Philadelphia positive acute lymphoblastic leukemia.

We describe a multiplex PCR assay for the detection of bcr-abl fusion mRNA in Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL). The assay provides a quick method for screening p190 (e1:a2) and p210 (b2:a2 or b3:a2) bcr-abl mRNAs simultaneously. The assay proves to be highly sensitive with detection of as little as one positive bcr-abl-expressing cell in a background of 10(5) negative bcr-abl cells. Bone marrow and peripheral blood specimens from six patients were in total accordance when run by multiplex PCR and by the single primer PCR approach. The multiplex bcr-abl assay may prove to be highly useful for screening newly diagnosed patients with ALL for the bcr-abl fusion transcript and in following the course of disease during therapy.

Base Sequence↗

Genetic identification and detection of human pathogenic Rhizopus species, a major mucormycosis agent, by multiplex PCR based on internal transcribed spacer region of rRNA gene.

BACKGROUND: Mucormycosis is an invasive opportunistic infection caused by fungi belonging to the order Mucorales. Due to the lack of laboratory tests, the diagnosis of mucormycosis is notoriously difficult. Added with its rapid progression as well as the debilitated state of the patients who contract the disease, mortality is extremely high. OBJECTIVE: The goal of this study was to genetically identify human pathogenic Rhizopus species, a major mucormycosis agent, by the internal transcribed spacer (ITS) region of rRNA gene. METHODS: Primers were designed to identify five Rhizopus species known to cause human disease by multiplex PCR. PCR was done not only with test strains and clinical isolates, but also with clinical samples from cutaneous mucormycosis patients. Sporangiospore morphology was observed by scanning electron microscopy to confirm the correlation of phenotypic and genotypic features. RESULTS: Multiplex PCR identified five Rhizopus species including Rhizopus oryzae, where R. azygosporus could only be distinguished from R. microsporus by certain polymorphisms that were present in its sequence. When this multiplex PCR was applied to clinical samples from three mucormycosis patients (paraffin sections from all and sera from one patient), Rhizopus DNA corresponding to the isolated pathogens were specifically detected. CONCLUSION: While fungal DNA detection from clinical samples is a rigorously studied area, this is the first report to genetically identify and detect Rhizopus species from human mucormycosis specimens. This may expand the possibility of this multiplex PCR system not only to identify isolated fungi, but also as a screening method for visceral mucormycosis.

Adolescent↗

Rapid screening of T-cell receptor (TCR) variable gene usage by multiplex PCR: application for assessment of clonal composition.

The selection of various T-cell receptor (TCR) gene families and complex rearrangements during intra-thymic differentiation provide the basis for the expression of antigen specificity by mature T cells. TCR beta variable (TCRBV) transcripts can be identified by RT-PCR, but multiple reactions are required to detect all genes of the TCRBV subfamilies. We describe here a multiplex PCR method that amplifies 46 functional genes comparing 23 TCRBV families in 5 reactions where each reaction contains 4 to 7 specific primers together with a single fluorescence-tagged TCR beta constant region primer. Between 8 and 10 distinct subtypes within each of the 23 TCRBV families can be identified by analysis of the CDR3 length. Multiplex PCR products isolated from agarose gels can be subjected to direct sequencing for confirmation and definitive clonotyping if necessary. The data illustrated here show that the multiplex PCR technique is useful for screening TCRBV usage and can be easily adapted for analysis of clonal composition in T-cell populations.

Cell Line↗

Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers.

Integration of human papillomavirus (HPV) DNA occurs early in cancer development and is an important event in malignant transformation of cervical cancer. Integration of HPVs preferentially disrupts or deletes the E2 open reading frame, which results in the loss of its expression. The preferential disruption of the E2 gene causes the absence of the E2 gene sequences in the PCR product following integration. Twenty-two carcinomas positive for HPV type 16 (HPV-16) DNA were first tested for the disruption of the E2 gene by PCR. A specific fragment of the E2 gene was not amplified in 10 cases, suggesting integration of HPV DNA into the host genome. Next, multiplex PCR for the HPV E2 and E6 genes was carried out in the remaining 12 cases. Copy numbers of both genes should be equivalent in episomal forms, while the E2 gene copy number will be smaller than that for E6 following the preferential disruption of the E2 gene in concomitant forms. Although relative ratios of HPV E2 to E6 PCR products (E2/E6 ratios) ranged from 1.40 to 2.34 in 10 of 12 cases, multiplex PCR products from 2 cases displayed extremely low ratios of 0.69 and 0.61. Southern blot hybridization with an HPV-16 probe revealed that only in these two cases was both episomal and integrated HPV DNA being carried simultaneously. Thus, multiplex PCR for the E2 and E6 genes of HPV-16 DNA following PCR for the E2 gene can distinguish the pure episomal form from a mixed form of episomal and integrated HPV DNA. Clinical application of this technique will help researchers to understand the implication of the integration of HPV DNA for cervical carcinogenesis and cervical cancer progression.

Base Sequence↗

Development and evaluation of a Mycobacterium avium subspecies paratuberculosis (MAP) specific multiplex PCR assay.

There are specificity questions inherent in most of the current PCR systems that amplify the MAP IS900 sequence as an indicator for Mycobacterium avium subspecies paratuberculosis (MAP) presence due to false positives associated with IS900-like sequences that exists in other Mycobacterium species besides MAP. We developed a multiplex PCR system designed to enhance specificity for MAP detection in a single PCR reaction. This PCR assay co-amplifies the mycobacterium species 16S rRNA gene, MAP IS900 and f57 sequences. The assay incorporates an internal PCR amplification control (IC) template system enabling PCR amplification conditions to be assessed in each reaction. The assay's specificity was confirmed by testing 10 isolates of MAP and 59 isolates of other bacterial species. In parallel we also applied on the same samples, one of the established nested PCR systems that targets only the IS900 sequence. The multiplex PCR assay was highly specific for MAP, the nested PCR system was also positive with several other mycobacterium species. In this context, we report for the first time false positive IS900 PCR signals in Mycobacterium chelonae, Mycobacterium terrae and Mycobacterium xenopi strains associated with one of the established PCR systems widely used for MAP detection. Finally, the potential application of this multiplex PCR system in milk analysis is demonstrated through analysis of raw milk samples artificially contaminated with MAP.

Animals↗

Multiplex PCR: rapid DNA cycling in a conventional thermal cycler.

Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more target sequences are simultaneously amplified in the same reaction. In the present study we investigated the limits to which the duration of multiplex PCR steps can be shortened using the thermal cycler Gene Amp PCR system 9600 (Perkin Elmer, Oak Brook, IL). The present multiplex PCR assay simultaneously detects five different herpes viruses (HSV-1, HSV-2, VZV, CMV, and EBV) and assesses sample suitability in a single amplification round of 40 cycles. It appears that when six target sequences are simultaneously amplified in multiplex PCR, extension time is a critical parameter. Using a PCR protocol of 0 sec at 95 degrees C, 0 sec at 60 degrees C, and 0 sec at 74 degrees C with Platinum Taq DNA polymerase (Life Technologies, Gaithersburg, MD), we were able to reduce the total cycling time of the multiplex PCR assay to as little as 55 min, without affecting the yield of PCR products or the specificity of the assay. It may be necessary to optimize each specific apparatus and template, but any such optimization would be trivial.

DNA↗

Development and amplification of multiple co-dominant genetic markers from single spores of arbuscular mycorrhizal fungi by nested multiplex PCR.

Multiple co-dominant genetic markers from single spores of the arbuscular mycorrhizal (AM) fungi Glomus mosseae, Glomus caledonium, and Glomus geosporum were amplified by nested multiplex PCR using a combination of primers for simultaneous amplification of five loci in one PCR. Subsequently, each marker was amplified separately in nested PCR using specific primers. Polymorphic loci within the three putative single copy genes GmFOX2, GmTOR2, and GmGIN1 were characterized by sequencing and single strand conformation polymorphisms (SSCP). Primers specific for the LSU rDNA D2 region were included in the multiplex PCR to ensure correct identification of the Glomus spp. spores. Single AM fungal spores were characterized as multilocus genotypes by combining alleles of each amplified locus. Only one copy of each putative single copy gene could be amplified from each spore, indicating that spores are homokaryotic. All isolates of G. mosseae had unique genotypes. The amplification of multiple co-dominant genetic markers from single spores by the nested multiplex PCR approach provides an important tool for future studies of AM fungi population genetics and evolution.

Alleles↗

Multiplex PCR assay to aid in the identification of the highly transmissible Mycobacterium tuberculosis strain CDC1551.

SETTING: Mycobacterium tuberculosis strain CDC1551 outbreak area in Tennessee and Kentucky and selected locations in the USA. OBJECTIVE: Develop a PCR assay to distinguish the highly transmissible CDC1551 from strains which have similar 4-band IS6110 fingerprints. DESIGN: Compare the IS6110 insertion sites in CDC1551 with those in 10 isolates which have similar 4-band IS6110 fingerprints. Utilize unique characteristics of insertion sites in CDC1551 to design a multiplex PCR to identify this strain. RESULTS: A multiplex PCR was developed which targets an IS6110 insertion conserved in most IS6110 low copy number strains and a deletion within the direct repeat region adjacent to an IS6110 insertion. Of 139 isolates with similar 4-band fingerprints, the CDC1551 PCR pattern was generated by only the 14 outbreak associated isolates. Of 154 isolates with different fingerprints, only four generated the CDC1551 pattern and these could be distinguished from CDC1551 by their IS6110 fingerprint. CONCLUSIONS: The multiplex PCR used in conjunction with the IS6110 fingerprint should be a useful tool to aid in the continued surveillance of the outbreak area and follow the spread of this highly transmissible strain of M. tuberculosis.

Bacterial Typing Techniques↗

Direct detection and identification of Pseudomonas aeruginosa in clinical samples such as skin biopsy specimens and expectorations by multiplex PCR based on two outer membrane lipoprotein genes, oprI and oprL.

A multiplex PCR test based on the simultaneous amplification of two lipoprotein genes, oprI and oprL, was designed and evaluated for its ability to directly detect fluorescent pseudomonads (amplification of oprI open reading frame, 249 bp) and Pseudomonas aeruginosa (amplification of oprL open reading frame, 504 bp) in clinical material. A collection of reference strains including 20 different species of fluorescent pseudomonads was tested. Positive PCR results for both genes were observed only for P. aeruginosa isolates (n = 150), including strains of clinical and environmental origin, while only one gene, oprI, was amplified from the other fluorescent pseudomonads. All other bacteria tested (n = 15) were negative by the amplification test. The lower detection level for P. aeruginosa was estimated to be 10(2) cells/ml. Preliminary evaluation on testing skin biopsy specimens from patients with burns (n = 14) and sputum samples from cystic fibrosis patients (n = 49) and other patients (n = 19) showed 100% sensitivity and 74% specificity in comparison with culture. This multiplex PCR assay appears promising for the rapid and sensitive detection of P. aeruginosa in clinical specimens. Further evaluation of its specificity in longitudinal clinical studies is warranted.

Bacterial Outer Membrane Proteins↗

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens.

AIMS: The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. METHODS AND RESULTS: The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. CONCLUSIONS: The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.

ADP Ribose Transferases↗

Development and evaluation of a multiplex PCR assay for rapid detection of toxigenic Vibrio cholerae O1 and O139.

A multiplex polymerase chain reaction assay was developed for concurrent detection of rfb sequences specific for the O1 and the O139 serogroups of Vibrio cholerae and for ctxA specific sequences. The multiplex PCR assay was found to be highly specific and sensitive and was capable of detecting 65 cfu and 200 cfu per assay of V. cholerae O1 and O139, respectively. Evaluation of the multiplex PCR assay using 121 stool samples from patients admitted to the Infectious Diseases Hospital, Calcutta, showed the assay to be 100% sensitive and 95.2% specific when the culture method was taken as the standard. In addition to the 38 PCR positive stool samples, an additional four samples which were negative by culture method but positive by PCR assay belonged to the O139 serogroup. All the 38 stool samples positive for either O1 or O139 serogroup by PCR assay were also positive for the ctxA amplicon indicating that the O1 and O139 V. cholerae strains have the genetic potential of producing cholera toxin.

Bacterial Proteins↗

Multiplex PCR for identification of methicillin-resistant staphylococci in the clinical laboratory.

A multiplex PCR assay for detection of the staphylococcal mecA gene (the structural gene for penicillin-binding protein 2a) was compared with agar dilution and disk diffusion susceptibility test methods for identifying methicillin resistance. The multiplex PCR assay combined two primer sets (mecA and 16S rRNA) in a single reaction. A total of 500 staphylococcal isolates (228 isolates of Staphylococcus aureus and 272 isolates of coagulase-negative staphylococci) from clinical specimens were studied. For S. aureus, 40 of 40 mecA-positive isolates and 4 of 188 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 100 and 98%, respectively). In 3 of 4 discordant isolates, resistance was due to hyperproduction of beta-lactamase. For coagulase-negative staphylococci, 148 of 159 mecA-positive isolates and 0 of 113 mecA-negative isolates were oxacillin resistant (positive and negative predictive values of 93 and 100%, respectively). Twenty-six isolates were categorized as indeterminate because of the absence of a detectable 16S rRNA product. Four of these 26 isolates contained mecA when retested. The assay is designed to be incorporated into the work flow of the clinical microbiology laboratory and allows for the identification of intrinsic resistance in a timely and reliable manner.

Base Sequence↗

Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products.

In this paper we report that the inclusion of heat-resistant RecA protein from a thermophilic bacteria, Thermus thermophilus, and its cofactor (ATP) in PCR effectively eliminates non-specific PCR products. The effect of RecA protein, which catalyzes pairing between homologous DNA molecules with great fidelity in genetic recombination, is due to its promotion of precise priming in PCR (i.e. priming at sites where the primer sequence is completely complementary to that of the target sequence). In addition, the RecA protein substantially reduces the primer concentration required for PCR. These experimental results have led to the realization of multiplex PCR, which involves PCR for multiple sites in the same reaction mixture. We were able to successfully perform multiplex PCR with over a dozen reactions without affecting the amplification pattern of the PCR products.

Base Pair Mismatch↗

Detection and characterization of verotoxigenic Escherichia coli by a VTEC/EHEC multiplex PCR in porcine faeces and pig carcass swabs.

To determine whether pigs and pig carcasses are a reservoir of verotoxigenic-producing Escherichia coli, a sensitive and rapid isolation method was developed. A VTEC/EHEC multiplex PCR method was developed to test all samples taken at the farm (n=289) and at the slaughterhouse (n=233). From the 137 positive VTEC/EHEC multiplex PCR farm samples, 54 strains were isolated that carried one virulence gene. Of these, thirty-one strains carried the vt2e variant of the vt2 gene, which causes oedemic disease in young pigs, four strains contained the hlyA gene and 19 the eaeA gene. All strains isolated were further identified with an enterotoxigenic E. coli (ETEC) multiplex PCR developed to detect the LTI, STIa, STII and vt2 genes in a single reaction. One-third of the vt2e-positive strains also contained the STIa gene. A small fraction of the 132 carcasses tested were found positive for VTEC or the E. coli O157 serotype. We conclude that none of the isolated strains carried a combination of virulence genes indicative of potential human pathogenic characteristics.

Abattoirs↗

Multiplex PCR for the detection of tetracycline resistant genes.

Specific primer pairs were selected for the PCR amplification of 14 tetracycline resistant genes commonly found in Gram positive and Gram negative organisms. Combinations of primer pairs were used in multiplex PCR reactions to detect specific groups of tet genes as follows; Group I tet (B), tet (C), tet (D); Group II tet (A), tet (E), tet (G); Group III tet (K), tet (L), tet (M), tet (O), tet (S); Group IV tetA (P), tet (Q), tet (X). To test the multiplex PCR, Groups I and II were used on 25 clinical isolates of Salmonella enterica serovar Typhimurium DT104. Group III primers were used to investigate 19 clinical isolates of methicillin-resistant Staphylococcus aureus. Multiplex PCR should result in significant savings in terms of labour and cost in analysis of a large number of strains when compared with using an individual PCR for targeting each gene. It may also be a useful method to differentiate the types of tetracycline resistance when used as an additional marker for the purpose of outbreak investigation and surveillance.

DNA Fingerprinting↗

[Multiplex PCR for the diagnostic detection of Coxiella burnetii in cow's milk].

A multiplex PCR based assay was developed for the highly sensitive and specific detection of Coxiella (C.) burnetii in cow's milk. The assay simultaneously amplifies a diagnostic target within the C. burnetii IS1111 sequence and a control target within the bovine CD18 gene. The internal PCR amplification control allows the discrimination of false negative results (single tube reaction failures) from negative results due to true absence of target sequences. In order to maximize the sensitivity of the assay, a sample preparation method including a centrifugation step to concentrate the bacterium was developed. In milk samples artificially contaminated with serial dilutions of C. burnetii, about four particles per ml could reproducibly be detected. The sensitivities of both assays, multiplex PCR and PCR with only a single pair of primers ('simplex' PCR), were observed to be similar.

Animals↗

Detection and typing of vancomycin-resistance genes of enterococci from clinical and nosocomial surveillance specimens by multiplex PCR.

Ninety-three clinical isolates of vancomycin-resistant enterococci (VRE) collected from nine hospitals in Taiwan were examined for the presence of vanA, vanB, vanC1, or vanC2/vanC3 genes by a multiplex PCR. Forty-seven of these VRE isolates were vanA positive, 1 contained both vanC1 and vanA, 40 harboured vanB, 2 were vanC1, and 3 were identified to be vanC2/vanC3. Twenty-four vanA isolates were sensitive to teicoplanin and thus did not have a typical VanA phenotype. Five isolates with the VanC phenotype harboured vanB. None of the 40 clinically isolated vancomycin-susceptible E. faecium or E. faecalis and the vancomycin-resistant Leuconostoc and Pediococcus isolates were positive for any of the van genes. While performing nosocomial surveillance, VRE were isolated from 47 of 467 rectal swabs by culture. Compared with the conventional culture method, the sensitivity and specificity of the multiplex PCR for detecting and identifying vancomycin-resistance genes in enterococci directly from culture-positive broth were 97.9% and 100%, respectively. The results suggest that genotypic characterization of vancomycin-resistance is necessary for all clinical VRE isolates and that the multiplex PCR assay can be an alternative method for this purpose.

Bacterial Proteins↗