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PubMed1998

Application of multiplex PCR for detection of non-O157 verocytotoxin-producing Escherichia coli in bloody stools: identification of serogroups O26 and O111.

Primers were designed to amplify sequences of verocytotoxin genes and eaeA genes of Escherichia coli O26:H11, O111:H8, and O157:H7 in a multiplex PCR assay. This assay successfully detected E. coli O26:H11 in bloody stool specimens in which other enteric pathogens were not detected by culture-based methods. Rapid assays to detect non-O157:H7 verocytotoxin-producing E. coli is important to improve methods for the etiologic diagnosis of hemorrhagic colitis.

Adhesins, Bacterial↗
PubMed2000

Multiplex PCR assays for the detection of clinically relevant antibiotic resistance genes in staphylococci isolated from patients infected after cardiac surgery. The ESPRIT Trial.

Multiresistant staphylococci (82 Staphylococcus aureus and 114 coagulase-negative staphylococci) were characterized by testing with rapid multiplex polymerase chain reaction (PCR) assays for species identification and detection of associated antibiotic resistance genes. These 196 staphylococci were isolated from 149 adult patients who developed wound infection after elective coronary artery bypass grafts and/or valve surgery. The multiplex PCR assays allowed identification of the most common staphylococcal species with S. aureus- and Staphylococcus epidermidis-specific primers as well as the detection of the erythromycin resistance genes ermA, ermB, ermC and msrA, the aminoglycoside resistance gene aac(6')-aph(2"), the oxacillin resistance gene mecA and the penicillin resistance gene blaZ. There was a very good correlation between the genotypic analysis by PCR and the phenotype determined by standard methods of susceptibility testing and identification of staphylococcal species: 100% for erythromycin resistance, 98.0% for gentamicin resistance, 99.0% for oxacillin resistance, 100% for penicillin resistance and 100% for S. aureus and S. epidermidis species identification. This study suggests that the incidence and distribution of the tested clinically relevant antibiotic resistance genes in staphylococci associated with infections after cardiac surgery do not differ from those in strains from other infections. These multiplex PCR assays may be used as diagnostic tools to replace or complement standard methods of susceptibility testing and identification of staphylococci.

Adult↗
PubMed1998

Identification of deletions and insertions in the p53 gene using multiplex PCR and high-resolution fragment analysis: application to breast and ovarian tumors.

We have developed a simple and highly efficient method to detect deletions and insertions in the p53 gene. All 11 exons of the p53 gene were amplified along with a control sequence in four multiplex PCR reactions in the presence of fluorescein-labeled primers. The PCR products were resolved on an automated sequencing gel and the DNA fragments were detected by fluorescence. Using this method, we screened 7 DNA specimens from ovarian tumors, 19 from breast tumors, and 26 from normal breast tissues. No abnormality was found in any of the DNA samples extracted from the normal tissues. A 19 base pair deletion in exon 5 of the p53 gene was detected in one ovarian tumor. Insertions were identified in two breast and two ovarian tumors. The insertions were identical in 3 of these tumors and consisted of a 16 bp repeat within intron 3 of the p53 gene. It appears that the insertion within intron 3 may represent a hot spot for duplication of the normal sequence at that site.

Breast Neoplasms↗
PubMed2002

Production of biogenic amines and divercin V41 in cold smoked salmon inoculated with Carnobacterium divergens V41, and specific detection of this strain by multiplex-PCR.

AIMS: The objective of this study was to determine the technological behaviour (implantation and biogenic amines production) of Carnobacterium divergens V41, an anti-Listeria bacteriocin producer (divercin V41), after inoculation in cold smoked salmon (CSS). METHODS AND RESULTS: Implantation of the strain was followed by multiplex-PCR during 27 days of storage at 4 degrees C, and biogenic amines were quantified by HPLC. It was found that the strain was able to develop quite well in CSS among lactic wild flora. Divercin V41 (400 AU ml-1) was produced in CSS, and the biogenic amine content was not modified by inoculation of the bacteria. CONCLUSIONS: Carnobacterium divergens V41 is a safe, interesting, bioprotective agent. SIGNIFICANCE AND IMPACT OF THE STUDY: This strain could potentially be used for efficient prevention of L. monocytogenes growth in CSS.

Animals↗
PubMed2004

Practical implementation of a multiplex PCR for acute respiratory tract infections in children.

Molecular testing for acute respiratory infections (ARIs) has documented value but limited implementation due to questions that typically slow the acceptance of new tests. This study sought to address these questions and achieve implementation. Rhinovirus was added to a nested multiplex PCR (M-PCR), increasing its diagnostic yield. Over one winter, three hospital pediatric departments used the M-PCR to complement their direct fluorescent-antibody assay (DFA) for respiratory syncytial virus (RSV). Clinicians recorded "pretest probability estimates" (using continuous scales for various pathogen groups) for comparison with test results; treatments and test turnaround times were also recorded. Transnasal and throat swabs, with or without nasopharyngeal aspirate (NPA), were M-PCR tested. NPA-containing sample sets found to be RSV positive by DFA were not further tested. Single PCR for human metapneumovirus (hMPV) was performed retrospectively. Of 178 ARI episodes representing 172 patients, NPA was included in 97 sample sets; 54 (56%) were determined to be RSV positive. The other NPA-containing sample sets (n = 43) yielded 27 findings (63%), and the swab-only sets (n = 81) yielded 47 findings (58%); rhinovirus was found most often. Testing for hMPV yielded seven positive results. M-PCR median turnaround times were 4 days in swab-only samples and 5 days with NPA. Antibiotics were prescribed in 50 episodes, at rates similar for RSV and rhinovirus. Pretest probability estimates of a viral cause were lower in episodes caused by rhinovirus than in episodes caused by RSV. The hospitals continued to use M-PCR for NPA-containing samples found to be RSV negative by DFA. Test implementation is more likely with higher diagnostic yield and a protocol that reflects day-to-day clinical and laboratory operations.

Acute Disease↗
PubMed2005

Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the species in the Bacillus cereus group.

Five different enterotoxins and one emetic toxin of Bacillus cereus have been characterized. To amplify all of the enterotoxin and emetic-specific sequences of the species in the B. cereus group, a multiplex PCR with 12 primer pairs was established. In developing the assay method, a common terminal sequence at the 3' ends of all primers was chosen and a hot start Taq polymerase was used to overcome primer dimer formation. The assay was successfully applied to analyze the toxigenic potential of 162 food-poisoning and food-related strains. Results showed that there were 10 toxigenic patterns for all the test strains. All of the B. cereus strains carried at least one toxin gene. More than 70% of Bacillus mycoides strains carried no known toxin genes. The toxin profiles and toxin genes of B. mycoides strains were significantly different from B. cereus strains (P < 0.05), although the two species were closely related. The results suggest that many B. mycoides strains might be less prone to cause food poisoning. They also indicate the importance of detecting the toxin genes together with the detection of the species in the B. cereus group.

Bacillus cereus↗
PubMed2006

One-tube multiplex PCR method for rapid identification of mycobacterium tuberculosis.

A rapid, inexpensive, simple, and accurate multiplex polymerase chain reaction (PCR) was developed in a single tube for identification of Mycobacterium tuberculosis. Assessment of sensitivity and specificity of simple PCR was performed with 116 strains of M. tuberculosis complex (MTC) and 144 strains of nontuberculous mycobacteria (NTM) compared with the biochemical method. Specific amplification of KS4, MTC-specific DNA fragment, was found in 98% (114/116) of MTC and not detected in 99% (143/144) of NTM. Amplification of the mtp40 gene revealed 95% sensitivity (100/105 strains of M. tuberculosis) and 77% specificity (not found in 119/155 mycobacterial strains). A multiplex PCR method based on the combination of KS4- and mtp40-derived primers was used for identification of M. tuberculosis. Crude DNA from slow growing mycobacteria with cream rough colonies that showed both 768-bp amplified product for KS4 and 396-bp for mtp40 was identified as M. tuberculosis whereas that from MTC gave only the 768-bp product.

Base Sequence↗
PubMed2005

A PCR multiplex and database for forensic DNA identification of dogs.

Animal-derived trace evidence is a common finding at crime scenes and may provide an important link between victim(s) and suspect(s). A database of 558 dogs of pure and mixed breeds is described and analyzed with two PCR multiplexes of 17 microsatellites. Summary statistics (number of alleles, expected and observed heterozygosity and power of exclusion) are compared between breeds. Marked population substructure in dog breeds indicates significant inbreeding, and the use of a conservative theta value is recommended in likelihood calculations for determining the significance of a DNA match. Evidence is presented that the informativeness of the canine microsatellites, despite inbreeding, is comparable to the human CODIS loci. Two cases utilizing canine DNA typing, State of Washington v. Kenneth Leuluaialii and George Tuilefano and Crown v. Daniel McGowan, illustrate the potential of canine microsatellite markers for forensic investigations.

Animals↗
PubMed2004

[Multiplex PCR in clinical microbiology].

The introduction of molecular biology methods in clinical microbiology laboratories brings important insights to obtain sensitive and specific diagnoses as fast as possible. These methods are not intended for replacement but for complement of the already applied microbiologic methods. The integrated analyses of all of them is bringing to the most feasible and efficient results. Within the molecular techniques applied, polymerase chain reaction (PCR) has acquired a great diagnostic value, permitting the identification of etiologic agents and the fast and sensitive detection of their virulence and resistance genotypes. Since some years ago, the development of the so called multiplex PCRs has gained deep interest. Those are reactions that get the simultaneous single tube amplification of different target sequences, allowing the simultaneous detection and identification of various genes of interest. In the present article, the most relevant applications of multiplex PCR for clinical microbiology are summarized.

Bacterial Infections↗
PubMed2003

Genotypic analyses of Vibrio parahaemolyticus and development of a pandemic group-specific multiplex PCR assay.

A total of 54 Vibrio parahaemolyticus strains including pandemic O3:K6 strains and newly emerged O4:K68, O1:K25, O1:K26, and O1:K untypeable strains (collectively referred to as the "pandemic group") were examined for their pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) profiles and for the presence or absence of genetic marker DNA sequences, toxRS/new or orf8, that had been reported elsewhere to be specific for the pandemic group. Both PFGE and AP-PCR analyses indicated that all strains of the pandemic group formed a distinct genotypic cluster, suggesting that they originated from the same clone. In addition to the pandemic group, four O3:K6 strains that did not possess the thermostable direct hemolysin (tdh) gene also belonged to this cluster and possessed the toxRS/new sequence. However, three O3:K6 strains that clearly belonged to the pandemic group by PFGE and AP-PCR did not possess the orf8 sequence. The evidence suggests that neither the toxRS/new nor the orf8 sequence is a reliable gene marker for definite identification of the pandemic group. We therefore developed a novel multiplex PCR assay specific for the pandemic group. The assay successfully distinguished pandemic group strains from other V. parahaemolyticus strains by yielding two distinct PCR products for tdh (263 bp) and the toxRS/new sequence (651 bp).

Bacterial Proteins↗
PubMed2002

The multiplex-PCR-based detection and genotyping of diarrhoeagenic Escherichia coli in diarrhoeal stools.

In several hospitals in Beirut, Lebanon, 77 isolates of Escherichia coli were successfully derived from the stools of patients with diarrhoeal diseases, by culture on MacConkey or MacConkey-sorbitol agar. When the isolates were screened, using a multiplex PCR, 14 (from 14 different patients) were each found positive for one of the various genes defining the enterotoxigenic (five), enteroinvasive (four), enteroaggregative (three) or enteropathogenic (two) groups. Genotyping of these 14 diarrhoeagenic isolates, by pulsed-field gel electrophoresis, indicated that all were genomically distinct with the exception of two of the enteroaggregative isolates (which were of the same genotype). The E. coli apparently involved in diarrhoeal disease in Beirut therefore belong to at least four different diarrhoeagenic groups and show strain variation within each group. Diarrhoea in the absence of diarrhoeagenic E. coli may be the result of infection with bacteria other than E. coli or viral or parasitic enteropathogens.

Adolescent↗
PubMed1996

Semi-multiplex PCR technique for screening of abundant transcripts during systematic sequencing of cDNA libraries.

The systematic sequencing of cDNA libraries is an efficient approach for the identification of new genes, but the presence of abundant mRNAs is often a major problem. This paper describes a very simple method of "semi-multiplex PCR" that allows specific identification of such abundant transcripts before DNA sequencing without using nonrepresentative subtracted libraries. The PCR utilizes a series of forward primers specific for abundant transcripts with a pair of universal primers used for template generation. cDNA clones corresponding to abundant mRNAs are then revealed by double bands in agarose gel.

Base Sequence↗
PubMed2003

Use of uniplex polymerase chain reaction & evaluation of multiplex PCR in the rapid diagnosis of viral retinitis.

BACKGROUND & OBJECTIVES: Polymerase chain reaction (PCR) has been known to be a rapid and accurate diagnostic test for causative viruses of viral retinitis, but cost is the limiting factor. In the present study an attempt was made to standardize a multiplex PCR (mPCR) on intraocular specimens from patients with viral retinitis for the detection of one or more viruses [herpes simplex virus (HSV), varicella zoster virus (VZV) or cytomegalovirus (CMV)] in order to reduce the period of time required for uniplex polymerase chain reaction (uPCR). METHODS: Using the uniplex PCR (uPCR) primers, a nested mPCR was developed and standardized for the simultaneous detection of HSV, VZV and CMV. mPCR and uPCRs were applied on 9 stored specimens and 38 prospective specimens obtained from patients with viral retinitis. RESULTS: The specificity and sensitivity of the mPCR were concordant with that of uPCRs. Clinical specificity and sensitivity of mPCR was further confirmed by the detection of the same herpes viral DNA on the 9 stored specimens. Of the 38 specimens collected prospectively, mPCR detected HSV in 3 (7.9%), VZV in 9 (23.7%), CMV in 5 (13.2%) and both VZV and CMV in 2 (5.3%). Co-infections of two viruses were found in 7 (14.89%) of the 47 specimens. INTERPRETATION & CONCLUSION: mPCR is a rapid, specific and sensitive diagnostic tool in viral retinitis. Compared to uPCR, mPCR is less time-consuming and cost effective.

Cytomegalovirus↗
PubMed1993

[Rapid screening of the Duchenne muscular dystrophy gene deletion by two multiplex PCR].

Duchenne muscular dystrophy (DMD) is one of the most common lethal X-linked disorders. Partial intragenic deletions account for up to 60% of DMD deletions, and approximately one-third of all cases arise via new mutations. In this paper, two sets of primers were used for two multiplex PCR screenings of DMD gene deletions. The first set consists of 5 pairs of primers which amplify 5 exons with higher deletion frequencies, and the second set includes other 4 pairs of primers. Fifty-one DMD samples were screened by the first set, and then those not found to have deletions were screened by the second set. In total, 25 deletions were identified in the Fifty-one DMD samples: That is, about 49% of all cases studied were found to have deletions.

Base Sequence↗
PubMed2003

Multiplex-PCR and PCR-RFLP assays to monitor water quality against pathogenic bacteria.

In this work we developed and optimized two molecular-based approaches to monitor rapidly, sensitively and specifically bacterial pathogens from three different genera, Escherichia coli, Pseudomonas aeruginosa, and Salmonella spp., directly in waters. To achieve this aim, firstly a multiplex-PCR assay (M-PCR) was optimized using a primer pair specific for each pathogen. Secondly, as a molecular confirmatory test after isolation of the pathogens by classical microbiological methods, PCR-RFLP of their amplified 16S rDNA genes was performed. It was observed from the results that the developed M-PCR assay has significant impact on the ability to detect sensitively, rapidly and specifically the three pathogens directly in water within a short time (5 h from sampling to obtain final results), therefore it represents a considerable advancement over other known more time-consuming and less-sensitive methods for identification and characterization of these kinds of pathogens.

DNA, Bacterial↗
PubMed1996

Simultaneous analysis of bovine kappa-casein and BLAD alleles by multiplex PCR followed by parallel digestion with two restriction enzymes.

An improved and simplified method allowing simultaneous genetic typing of kappa-casein and CD 18 (bovine leucocyte adhesion deficiency; BLAD) loci has been developed. The method is based on the simultaneous amplification of fragments of the two groups of alleles by multiplex PCR, and on a concurrent, parallel digestion of the products by two restriction enzymes (PstI and HaeIII) in the same incubation buffer. Digestion with PstI distinguishes kappa-casein A and B alleles and does not cut within any of the BLAD alleles, while digestion with HaeIII allows the differentiation between normal and mutant allele variants of the CD18 locus. All combinations of the known mutants of the two alleles, characterized to the regions amplified and resulting in phenotypic effect, could be detected by electrophoretic separation performed on the same agarose gel owing to the vast differences in the length of the restriction fragments.

Alleles↗
PubMed2006

[The tk gene mutation analysis in WTK1 cell line by multiplex PCR].

OBJECTIVE: To explore the tk gene mutation spectrum in spontaneous and induced WTK1 mutants. METHODS: After exposure to methyl methanesulfonate (MMS), mitomycin (MMC) or sodiam azide (NaN3), spontaneously-arising and induced mutants of WTK1 cell line were selected. The spectrum and hotspot of tk gene mutation were analyzed by multiplex PCR. RESULTS: Three chemicals used in this study induced tk mutation frequency (MF) to increase significantly in a dose-dependent manner; most of the analyzed mutants had lost exon 4 and exon 5-7. CONCLUSION: Three chemicals have mutagenic effect on WTK1 cell line, and obvious hotspot exists in tk gene mutation.

Cells, Cultured↗