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Results for “Multiplex PCR”

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At least 343 records · Page 19Linked to original sources

PubMed1998

Detection of HCV and GBV-C/HGV infection by multiplex PCR in plasma samples of transfused subjects.

A multiplex polymerase chain reaction (PCR) was applied to clinical samples for simultaneous detection of hepatitis C virus (HCV) and GBV-C/HGV genome. With both RNA viruses, the amplification was performed with primers of the 5' UTR region starting from the single viral RNA reverse transcripted (cDNA) with random hexanucleotide primer mix. GBV-C/HGV RNA was detected in plasma sample of seven out of 50 transfused patients (14%). The multiplex PCR demonstrated a sensitivity up to 7.8 x 10(2) copies/ml respectively for GBV-C/HGV and HCV RNA in plasma samples of 5/50 patients with GBV-C/HGV/HCV co-infection and in patients with HCV (27/50) or GBV-C/HGV infection alone (2/50).

Blood Transfusion↗

PubMed2003

Subtyping of foodborne and environmental isolates of Escherichia coli by multiplex-PCR, rep-PCR, PFGE, ribotyping and AFLP.

A total of 54 isolates were characterized by multiplex-PCR for toxin genes and genotyped using several DNA fingerprinting methods: using repetitive extragenic palindromes (REP) and Box primers (rep-PCR), amplified fragment length polymorphism (AFLP), pulsed-field gel electrophoresis (PFGE) and ribotyping. The known-pathogenic strains tested were from food and clinical samples (34 strains) and included serovars O157:H7, O111:H8, O111:H11, O91:H21 and O55:H7. Two type cultures, Escherichia coli K12 (ATCC 29425) and DUP-101 (ATCC 51739), were included as known non-pathogenic strains and an additional 17 previously unclassified isolates from animal fecal samples. Comparisons of genomic DNA fingerprint patterns using unweighted pair group method with arithmetic averages (UPGMA) cluster analysis of Jaccard similarity indices indicated that all methods tested showed a greater similarity between the E. coli O157:H7 strains than to other isolates. On the basis of these studies, we propose that AFLP, REP-PCR, Box-PCR and ribotyping techniques can all be used for discriminating O157:H7 isolates and are preferred for large-scale screening because of the speed and ease of the methods. The PFGE method is the best to discriminate between subtypes of O157:H7 associated with specific outbreak investigations; however, it is more time consuming and unnecessary if subtyping is not required. There are differences between the dendrograms generated from each method and the relationship between the other strains analyzed. However, the fingerprint profiles of the O157:H7 isolates were virtually identical using REP-PCR and Box-PCR enabling easy distinction of the group. Thus, these typing methods have the potential to aid investigators in identifying the source of an outbreak to prevent or control further spread of E. coli O157:H7.

Bacterial Toxins↗

PubMed2001

Multiplex PCR protocol for the diagnosis of staphylococcal infection.

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.

Blood↗

PubMed2004

Rapid diagnosis of bacterial meningitis by using multiplex PCR and real time PCR.

BACKGROUND: The purpose of the present study was to improve a method for a rapid identification of bacteria in bacterial meningitis by using multiplex polymerase chain reaction (PCR). METHODS: Ten species of bacteria which cause meningitis in children were investigated, and cerebrospinal fluid from patients with purulent meningitis was studied. The ribosomal RNA genes of bacteria are essential, and are highly conserved in the bacterial kingdoms with consensus region. The 23S rRNA region shows a larger variation among species than in the 16S rRNA region. The authors set primers in the universal region and specific region of 23S rRNA, then amplified these regions by multiplex PCR and real-time PCR. RESULTS: All species of bacteria showed one band by PCR using universal primer. Haemophilus influenzae and Streptococcus pneumoniae showed two bands by multiplex PCR using a combination of universal primers and specific primers. The authors detected H. influenzae within 15 min by using real-time PCR. CONCLUSION: It was possible to identify clinically significant bacterial species in cerebrospinal fluid by multiplex PCR, and to identify H. influenzae by real-time PCR within a short period.

Cerebrospinal Fluid↗

PubMed2004

Multiplex PCR for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila in community-acquired pneumonia.

A multiplex polymerase chain reaction (PCR) was developed for the simultaneous detection of Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella pneumophila. Oligonucleotide primers for the amplification of the DNA of these three organisms were optimized for use in combination in the same reaction. PCR products were detected by the Micro-Chip Electrophoresis Analysis System. Clinical samples were obtained from 208 community-acquired pneumonia (CAP) patients who were participants in a multicenter CAP surveillance study performed at seven medical schools and their affiliate hospitals in Japan. No significant differences in the sensitivity of each primer set were observed when tested in both the multiplex and monoplex PCR assays. Our multiplex PCR was able to reliably detect 10 copies/100 microl of each of the three pathogen DNAs. Of the panel of 208 samples, 14 of 15 C. pneumoniae, 10 of 10 M. pneumoniae, eight of eight L. pneumophila and 165 of 176 negative samples were correctly identified. Eleven cases who were the multiplex PCR positive and conventional method negative were observed. The PCR findings were of possible significance in at least four of these patients. Our multiplex PCR assay could potentially be used as a diagnostic and epidemiological tool. Further prospective studies are needed to establish its clinical usefulness.

Adolescent↗

PubMed1996

Using multiplex PCR amplification and typing kits for the analysis of DNA evidence in a serial killer case.

Analysis of DNA evidence in a serial killer case was performed using the AmpliType HLA-DQ alpha-, AmpliType PM-, and the GenePrint STR Multiplex System PCR Amplification Kits. In addition, a sex typing procedure using the X-Y homologous gene amelogenin was carried out. DNA profiles from a single hair with attached sheath material, recovered from underneath the seat cover of the suspect's car seat were compared with DNA profiles derived from reference head hairs from a homicide victim. From the evidentiary sample only 9 ng of human DNA could be recovered. In a sample, where the quantity of DNA becomes a critical issue a powerful route is the simultaneous amplification of several loci (multiplex PCR). This is the first report where commercially available multiplex PCR amplification and typing kits have been introduced for the analysis of DNA evidence in a serial killer case and the analysis has been admitted in court.

Adult↗

PubMed2002

Simultaneous detection of measles virus, rubella virus, and parvovirus B19 by using multiplex PCR.

We describe here a multiplex reverse transcription-PCR (RTMNPCR) assay designed to detect and differentiate measles virus, rubella virus, and parvovirus B19. Serial dilution experiments with vaccine strains that compared cell culture isolation of measles in B95 cells and rubella in RK13 cells showed sensitivity rates of 0.004 50% tissue culture infective dose (TCID(50)) for measles virus and 0.04 TCID(50) for rubella virus. This RTMNPCR can detect as few as 10 molecules for measles virus and rubella virus and one molecule for parvovirus B19 in dilution experiments with plasmids containing inserts of the primary reaction amplification products. Five pharyngeal exudates from measles patients and 2 of 15 cerebrospinal fluid samples from measles-related encephalitis were found to be positive for measles virus by this RTMNPCR. A total of 3 of 27 pharyngeal exudates from vaccinated children and 2 pharyngeal exudates, plus one urine sample from a case of congenital rubella syndrome, were found to be positive for rubella virus by RTMNPCR, whereas 16 of 19 sera from patients with erythema infectiosum were determined to be positive for parvovirus B19 by RTMNPCR. In view of these results, we can assess that this method is a useful tool in the diagnosis of these three viruses and could be used as an effective surveillance tool in measles eradication programs.

Child↗

PubMed2000

Simultaneous detection of hepatitis B, C, and G viral genomes by multiplex PCR method.

We established a multiplex polymerase chain reaction (PCR) method for simultaneous detection of hepatitis B, C, and G viral genomes. The levels of concordance with the data obtained by conventional single PCR method were 100% for single infection, 98 to 100% for double infections, and 92% for triple infections. This method is not only suited to rapid, large-scale epidemiological screening and clinical diagnosis of those virus infections occurring alone or in combination, but is also time- and cost-effective.

Cost-Benefit Analysis↗

PubMed2026

Improving quality control of microbial agri-inputs by confirming strain identity with an easy and low-cost PCR-multiplex: A study case with Azospirillum brasilense.

The first commercial product containing the Azospirillum brasilense elite strains Ab-V5 and Ab-V6 was launched in Brazil in 2009. These strains have demonstrated agronomic efficiency in grasses and in legume co-inoculation, accounting for approximately 43 million doses in 2024. Official identification of these strains is currently performed by rep-PCR, a reliable but time-consuming and laborious method. In this study, a multiplex PCR assay was developed for the simultaneous identification of Ab-V5 and Ab-V6 in a single reaction using strain-specific SNPs. Forward primers were designed so that the terminal nucleotide at the 3' end corresponded to a strain-specific SNP unique to each target strain. To further enhance specificity, artificial mismatches were introduced at the fourth nucleotide from the 3' end of the forward primers. SNPs were identified using Snippy based on genomic alignments between Ab-V5 and Ab-V6 and confirmed by local BLASTn against the genomes of other Azospirillum species. In the multiplex assay, simultaneous and specific amplification of both strains was observed in a single reaction, without non-specific amplification. Primer specificity was also experimentally evaluated against other A. brasilense strains (Ab-V1, Ab-V2, Ab-V4, Ab-V7, Ab-V8, and Sp7T), in silico against bacteria from different genera associated with agricultural inoculants, and in commercial inoculant samples containing Ab-V5 and Ab-V6. The results confirmed the high specificity of the primers for Ab-V5 and Ab-V6 and demonstrated that the assay was capable of identifying the strains in commercial inoculants. This assay facilitates inoculant quality control by enabling strain confirmation using a simple, rapid, and low-cost method.

Azospirillum↗

PubMed2004

Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish.

In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus. Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates. The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V. vulnificus. No amplification of other Vibrio spp. or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method. The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 10(3) V. vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate. This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples. A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method. Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons. The rapid, sensitive, and specific detection of clinically important pathogenic V. vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen.

Amides↗

PubMed2005

Development and evaluation of a multiplex PCR for differentiation of foot-and-mouth disease virus strains native to India.

A multiplex PCR (mPCR) for the differentiation of Indian FMDV serotypes, O, A, Asia 1 and C was developed and evaluated on 142 clinical and 39 cell culture samples. On the latter samples both the tests worked well with 100% efficiency, whereas on clinical samples mPCR had better efficiency than ELISA. The test was found to be specific for FMDV. The detection limit of the assay was varied among the serotypes; it was most sensitive on types A and Asia 1 and least sensitive on type C. The mPCR clearly identified the serotype and in some cases detected dual infections. The test is sensitive and reliable and can be used for serotyping of ELISA negative samples.

Animals↗

PubMed1995

Multiplex PCR for detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Genitourinary specimens.

We developed a multiplex PCR (M-PCR) assay for the simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. M-PCR employed C. trachomatis-specific primers KL1-KL2 and N. gonorrhoeae-specific primers HO1-HO3 and produced products of 241 and 390 bp, respectively. PCR products were easily detected by agarose gel electrophoresis and confirmed by Southern hybridization using labelled oligonucleotide probes. M-PCR had a sensitivity of 10 fg of C. trachomatis and N. gonorrhoeae DNA (equivalent to 1 to 2 genome copies). M-PCR detected the presence of C. trachomatis and N. gonorrhoeae DNA in 15 male urethral and 12 female endocervical specimens, 3 of which were positive for C. trachomatis, 18 of which were positive for N. gonorrhoeae and 6 of which were positive for both organisms. M-PCR was evaluated further by testing 200 male first void urine (FVU) specimens, of which 18 were positive by C. trachomatis PCR and Chlamydiazyme and 4 were positive by C. trachomatis PCR but negative by Chlamydiazyme. All 22 FVU specimens were positive by a confirmatory PCR using a second plasmid target and were positive by M-PCR. Ten of 11 men with cultures that were positive for N. gonorrhoeae had FVU specimens that were positive by both N. gonorrhoeae PCR and M-PCR. Two other men with negative N. gonorrhoeae urethral cultures had FVU specimens that were positive by N. gonorrhoeae PCR, by two confirmatory N. gonorrhoeae PCR assays using 165 rRNA and cytosine methyltransferase primers, and by M-PCR. The sensitivity of M-PCR for detecting C. trachomatis was 100% (22 of 22 specimens), compared with 81.8% (18 of 22 specimens) for enzyme immunoassay. Sensitivity of M-PCR for N. gonorrhoeae was 92.3% (12 of 13 specimens) compared with 84.6% (11 of 13 specimens) for urethral culture. The specificity of M-PCR was 100% for both C. trachomatis (178 of 13 specimens) and N. gonorrhoeae (187 of 187 specimens). M-PCR testing of FVU specimens provided a sensitive and noninvasive method for detecting C. trachomatis and N. gonorrhoeae infection in men.

Adolescent↗

PubMed1997

Multiplex PCR for diagnosis of AIDS-related central nervous system lymphoma and toxoplasmosis.

A nested multiplex PCR assay was designed for the simultaneous detection of Epstein-Barr virus and Toxoplasma gondii DNA from the cerebrospinal fluid of AIDS patients. T. gondii DNA was detected in 8 of 8 patients with Toxoplasma encephalitis and in 0 of 6 patients without toxoplasmosis, and Epstein-Barr virus DNA was found in 9 of 14 patients with central nervous system lymphoma and in 2 of 38 patients without disease.

AIDS-Related Opportunistic Infections↗

PubMed2003

Use of multiplex PCR to identify Staphylococcus aureus adhesins involved in human hematogenous infections.

We have developed a multiplex PCR procedure to determine the distribution of nine adhesin genes in Staphylococcus aureus isolates. Only genes encoding bone sialoprotein binding protein and fibronectin binding protein B were significantly associated with hematogenous osteomyelitis/arthritis and native-valve endocarditis, respectively, suggesting their involvement in hematogenous tissue infections.

Adhesins, Bacterial↗

PubMed2001

[Rapid detection of enterohemorrhagic Escherichia coli in food with multiplex PCR assay].

Rapid detection of enterohemorrhagic Escherichia coli (EHEC) in food was achieved by using multiplex PCR assay. The assay was designed to amplify the 1109, 302 and 228 bp regions of corresponding virulence genes eaeA, hlyAB, slt1 and/or slt2, by using specific primers for each one reaction. In 60 EHEC and other strains, the above three virulence gene sequences were co-amplified from DNA isolated from 12 EHEC O157:H7 strains, 1 O26:H11 strain and 1 O111:H8 strain; eaeA sequence was amplified from DNA isolated from 2 EAEC strains; slt1/2 sequence was amplified from DNA isolated from 1 VTEC strain. No specific amplification product was produced from other 43 non-EHEC strains. For the detection of EHEC in food, results could be obtained within 8 hrs, and the sensitivity of assay was less than or equal to 1.6 cfu/g(ml) of food. The method was applied to 126 food samples, and EHEC was detected in 3 samples.

Animals↗

PubMed2004

[Multiplex PCR for detecting genotypes of deletional alpha-thalassemia].

To investigate the clinical application of multiplex PCR in detecting genotypes of deletional alpha-thalassemia in South China and observe the distribution frequency of alpha-globin gene deletion, 145 patients with silent carrier, alpha thalassemia trait or HbH were identified by M-PCR and 1.2% agarose gel electrophoresis. There are 1.3, 1.6, 1.8 and 2.0 kb bands which indicate --(SEA), -alpha(4.2), alphaalpha and -alpha(3.7), respectively. The results showed that among 145 patients, 100 patients with --(SEA)/alphaalpha (68.9%), 15 with -alpha(3.7)/alphaalpha (10.3%), 8 with -alpha(4.2)/alphaalpha (5.52%), 2 with -alpha(3.7)/-alpha(4.2) (1.38%), 1 with -alpha(3.7)/-alpha(3.7) (0.69%), 1 with -alpha(4.2)/-alpha(4.2) (0.69%), 14 with --(SEA)/-alpha(3.7) (9.65%), 2 with --(SEA)/-alpha(4.2) (1.38%) were found. Two patients prenatal diagnosed were confirmed with Bart's hydrops fetuses. In conclusion, M-PCR analysis is a simple, rapid and accurate method for detection of alpha-thalassemia gene deletion. This technique is helpful in screening, carrier identification and prenatal diagnosis of deletional alpha-thalassemia.

Adolescent↗

PubMed2001

LightCycler multiplex PCR for the laboratory diagnosis of common viral infections of the central nervous system.

A conventional multiplex PCR assay that detects herpes simplex virus type 1 (HSV-1), HSV-2, varicella-zoster virus, and enteroviruses for the diagnosis of central nervous system infections was modified to be performed using the LightCycler system. The sensitivity of detection of each of the viruses using the LightCycler assay was compared to that of the conventional assay using external quality assessment material. The assays had equivalent sensitivities, but the LightCycler assay was more rapid, reduced the risk of contamination, and used an amplicon detection format that demonstrated greater discrimination than a gel electrophoresis method.

Central Nervous System Viral Diseases↗

PubMed2005

Development of a multiplex PCR assay for the identification of Staphylococcus aureus enterotoxigenic strains isolated from milk and dairy products.

A multiplex PCR for the simultaneous detection of Staphylococcus aureus 23S rRNA, the coagulase and thermonuclease genes as well as the enterotoxin genes sea, sec, sed, seg, seh, sei, sej, sel was developed. The method was used to determine the presence of enterotoxigenic types for 93 S. aureus strains isolated from milk and dairy products. The data obtained by mPCR resulted comparable to those obtained by immunoassay methods. In addition, the mPCR assays also amplified some se genes, whose toxins are undetectable by immunoassay. Multiplex amplification can be obtained starting from 1 pg of DNA, showing the excellent specificity and high sensitivity of the assay.

Animals↗