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PubMed2006

Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp.

Carbapenem resistance in Acinetobacter baumannii is a growing public health concern and is most often mediated by OXA carbapenemases. We describe a novel multiplex polymerase chain reaction (PCR) assay able to detect and distinguish alleles encoding three subgroups of acquired OXA carbapenemases (OXA-23-like, OXA-24-like and OXA-58-like) that are scattered in Acinetobacter spp., and a fourth subgroup, OXA-51-like, which appears to be intrinsic to Acinetobacter baumannii. Isolates belonging to two prevalent UK A. baumannii 'OXA' clones (OXA-23 clones 1 and 2) had alleles encoding both an intrinsic OXA-51-like and an acquired OXA-23 enzyme, whereas isolates of the 'SE clone' had only an intrinsic bla(OXA-51-like) allele. Genes encoding OXA-58 were detected (with bla(OXA-51-like)) in a cluster of related isolates from a single hospital. This simple assay will assist in monitoring the mechanisms responsible for carbapenem resistance in Acinetobacter spp.

Acinetobacter↗

PubMed2005

Differentiation of five strains of infectious bursal disease virus: development of a strain-specific multiplex PCR.

Infectious bursal disease virus (IBDV) is a major cause of disease problems in the poultry industry and vaccination has therefore been applied intensively to control the infection. The classical methods of detection and characterization of IBDV are by the use of immunodiffusion test and histopathology. Since these methods are laborious and have low specificity alternatives are needed. In the present study, we report the development of a strain-specific multiplex RT-PCR technique, which can detect and differentiate between field strains of IBDV and vaccine virus strains including a so-called hot vaccine strain widely used in the European poultry industry. The method, which is highly specific, fast and inexpensive, can be applied in all laboratories with basal PCR capabilities and equipment.

Animals↗

PubMed2000

Northeast Italy population data using multiplex PCR (HUMCD4, HUMTH01, HUMTPOX, and HUMCSF1P0) loci.

Allele and genotype frequencies for four short tandem repeat (STR) loci (HUMCD4, HUMTH01, HUMTPOX, and HUMCSF1P0) were determined in 100 unrelated individuals from Veneto (Northeast Italy). After a multiplex polymerase chain reaction (PCR)-amplification, semi-automatic DNA profiling was performed using an A.L.F.express DNA Sequencer. Conditions were optimized for the PCR co-amplification of these four STR loci and the quadruplex PCR was performed on various forensic DNA samples such as whole blood, blood-stains, teeth, and saliva from Caucasians living in the Northeast Italy. The distribution of the genotype frequencies showed no significant deviation from Hardy-Weinberg expectations in the sampled population. The combined Power of Discrimination (PD) of the quadruplex was 0.9999.

DNA Fingerprinting↗

PubMed2005

Real-time multiplex PCR assay to quantify hepatitis C virus RNA in peripheral blood mononuclear cells.

Ultrasensitive methods to measure very low levels of hepatitis C virus (HCV) RNA in biological samples may have diagnostic and prognostic significance and be useful to evaluate the response to antiviral treatment. A sensitive assay to quantify HCV RNA in peripheral blood mononuclear cells (PBMCs) was developed and validated using the iCycler iQ Detection System (Bio-Rad) coupled with TaqMan chemistry. HCV was co-amplified with the endogenous control glyceraldehyde-3-phosphate dehydrogenase in a multiplex reaction. Calculated PCR amplification efficiencies for both target and control genes were used in a mathematical model for relative quantitation of HCV RNA. A linear relationship between input RNA and C(T) values over 6 log dilutions was observed for both HCV- and GAPDH-specific products (R(2) > or = 0.99). As few as 1.5 IU/reaction could be detected, with high accuracy (CV < or= 3.94%) and reproducibility (CV < or = 2.20%). Quantitation of HCV RNA levels ranging from 10(3) to 10(7) IU/ml as measured in 47 plasma samples was highly correlated with values obtained by the COBAS Amplicor HCV Monitor test, v2.0 (Roche) (R(2) = 0.977). In conclusion, this assay provides an excellent tool to determine accurately HCV kinetics in PBMCs during antiviral therapy and to assess the long-term significance of different patterns of response to treatment.

5' Untranslated Regions↗

PubMed2003

Serial quantification of lymphoid and myeloid mixed chimerism using multiplex PCR amplification of short tandem repeat-markers predicts graft rejection and relapse, respectively, after allogeneic transplantation of CD34+ selected cells from peripheral blood.

We used multiplex amplification of nine microsatellite sequences (PCR-STR) to analyse chimerism in pure populations of T cells and neutrophils from peripheral blood from 40 patients submitted to an allogeneic transplant, 22 having received a T-cell depleted (TCD) peripheral blood graft by means of CD34(+) selection (allo-PBT/CD34(+)), and 18, an unmodified graft (allo-SCT; 13 allogeneic bone marrow transplants and five allo-PBT). T-cell mixed chimerism (TcMC) was observed in 16 of the 22 (72.3%) patients receiving an allo-PBT/CD34(+), but in only one of the 18 (5.5%) patients receiving an allo-SCT (P=0.0001). TcMC was transient (n=6), stable (n=7), and associated with poor haematopoietic engraftment (n=4). All patients with TcMC who developed graft failure had more than 30% of host T cells. Myeloid MC (MyMC) was observed in four (19%) allo-PBT/CD34(+) patients and in three (17%) allo-SCT patients (P=NS). Five out of seven (71%) patients with MyMC relapsed, all of them diagnosed with myeloid malignancies, as compared with two of the 20 (10%) patients with complete donor chimerism (P&<0.0001). In conclusion, TcMC appears in a significant number of allo-PBT/CD34(+) patients and may be associated with poor engraftment when the percentage of host T cells is >30%; likewise, MyMC appears in a small percentage of recipients of both allo-PBT/CD34(+) and allo-SCT patients, and is associated with leukaemia relapse in myeloid malignancies.

Adult↗

PubMed2004

Bacterial and viral DNA in periodontal disease: a study using multiplex PCR.

Recent studies have suggested an association between periodontal disease and the presence of Herpesviruses, in particular: Epstein-Barr virus (EBV) and Human Cytomegalovirus (CMV) (Contreras et al., 1999--Contreras et al., 2000--Slots et al., 2000--Ting et al., 2000). In the work reported in this paper, we use a multiplex Polymerase Chain Reaction (PCR) to compare the presence of Herpesviruses and putative bacterial pathogens in patients with periodontal disease and in healthy individuals. Direct detection of microorganisms with PCR is shown to offer significant advantages in terms of time, effort and cost. The study detected no statistically significant differences between the prevalence of EBV and CMV in patients and controls. The failure to replicate previous findings may be due to differences in the age composition and the geographical and social origins of the study groups. The study detected a significant excess of HSV-1 in periodontal patients. This suggests that the role of Herpesviruses in the pathogenesis of periodontal disease deserves further investigation. The bacterial assay confirmed the results of previous studies showing a strong association between periodontitis and the presence of A. actinomycetemcomitans, P. gingivalis and P. intermedia.

Actinobacillus Infections↗

PubMed2001

Rapid and simultaneous identification of body parts from the morphologically similar sharks Carcharhinus obscurus and Carcharhinus plumbeus (Carcharhinidae) using multiplex PCR.

Many commercially exploited carcharhinid sharks are difficult to identify to species owing to extensive morphological similarities. This problem is severely exacerbated when it comes to identifying detached shark fins, and the finless and headless shark carasses typically sold in markets. To assist in the acquisition of urgently needed conservation and management data on shark catch and trade, we have developed a highly streamlined approach based on multiplex polymerase chain reaction (PCR) that uses species-specific primers derived from nuclear ribosomal ITS2 sequences to achieve rapid species identification of shark body parts. Here we demonstrate the utility of this approach for identifying fins and flesh from two globally distributed, morphologically very similar carcharhinid sharks (Carcharhinus obscurus and Carcharhinus plumbeus) intensively targeted in fisheries worldwide, and often confused for each other even as whole animals. The assay is conducted in a 4-primer multiplex format that is structured to simultaneously achieve the following efficiency and cost-reduction objectives: it requires only a single-tube amplification reaction for species diagnosis, it incorporates an internal positive control to allow detection of false-negative results, and it is novel in that it allows species identification even when DNAs from two species are combined in the same tube during the PCR reaction. The latter innovation reduces the required effort for screening a set of unknown samples by 50%. The streamlined approach illustrated here should be amenable for use in a shark conservation and management context where large numbers of samples typically need to be screened; the approach shown may also provide a model for a rapid diagnostic method applicable to species identification in general.

Journal Article↗

PubMed2003

Multiplex PCR assay for Cylindrospermopsis raciborskii and cylindrospermopsin-producing cyanobacteria.

Water bodies are routinely monitored for the presence of potentially toxic cyanobacteria; however, the methodology for confirming toxicity is currently complex and expensive. Here we describe the application of gene-based technology to rapidly identify cylindrospermopsin-producing cyanobacteria, specifically, Cylindrospermopsis raciborskii. A multiplex polymerase chain reaction (PCR) test was developed that simultaneously identified polyketide synthase (pks) and peptide synthetase (ps) determinants associated with cylindrospermopsin production and distinguished C. raciborskii from other cylindrospermopsin-producing cyanobacteria of the species Anabaena bergii and Aphanizomenon ovalisporum, by targeting the rpoC1 gene. Twenty-one C. raciborskii, 5 A. bergii, 10 Aph. ovalisporum isolates and 3 environmental samples all yielded PCR results consistent with their toxicological status, as assessed by high-performance liquid chromatography coupled to mass spectrometry or matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and C. raciborskii was always correctly identified. The PCR test is a rapid, reliable, and economical way of assessing the toxic potential of cyanobacterial blooms formed by these organisms.

Alkaloids↗

PubMed2001

[Genetic studies of 13 loci in Guangdong Han population by two multiplex PCR systems and its forensic application].

OBJECTIVE: The genetic studies of 13 short tandem repeats(STRs) loci in two multiplex amplification systems were carried out on Chinese Han population in Guangdong. METHODS: DNA samples from 328 unrelated individuals were screened. The 13 loci were D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, TH01, TPOX and CSF1PO. The PCR products were analyzed and genotyped by ABI 377-96 Sequencer. RESULTS: The combined power of discrimination (DP) was 0.999999999999993 and the combined paternity of exclusion(PE) was 99.999%. These 13 loci met the Hardy-Weinberg expectations. CONCLUSION: The two multiplex amplification systems were very useful in forensic case investigation.

Asian People↗

PubMed1999

Rapid identification of Mycobacterium bovis BCG by the detection of the RD1 deletion using a multiplex PCR technique.

The BCG vaccine strain cannot, with confidence, be differentiated from other members of the Mycobacterium tuberculosis complex on phenotypic tests alone. Isolates from clinical sites not associated with vaccination may be confused with M. tuberculosis. A characteristic of BCG strains is the deletion of the genomic region RD1; detection of this forms the basis of a multiplex polymerase chain reaction (PCR) assay to distinguish BCG strains. In this study, 28 M. tuberculosis complex strains were analysed by the PCR assay. A DNA sequence displaying the characteristic deletion was detected in all eleven of the BCG strains tested and was not found in representatives of other members of the complex, including M. bovis. Thus, the assay affords a rapid, simple and effective method for the discrimination of the BCG vaccine strain from other members of the M. tuberculosis complex.

BCG Vaccine↗

PubMed2004

Development of a multiplex PCR for detection of avian adenovirus, avian reovirus, infectious bursal disease virus, and chicken anemia virus.

A multiplex polymerase chain reaction (mPCR) was developed and optimized for the simultaneous detection and differentiation of avian reovirus (ARV), avian adenovirus group I (AAV-I), infectious bursal disease virus (IBDV), and chicken anemia virus (CAV). Four sets of specific oligonucleotide primers were used in this test for ARV, AAV-I, IBDV, and CAV. The mPCR DNA products were visualized by gel electrophoresis and consisted of fragments of 365 bp for IBDV, 421 bp for AAV-I, 532 bp for ARV, and 676 bp for CAV. The mPCR assay developed in this study was found to be sensitive and specific. Detection of PCR-amplified DNA products was 100 pg for both CAV and IBDV, and 10pg for both ARV and AAV-I and this mPCR did not amplify nucleic acids from the other avian pathogens tested. The mPCR demonstrated similar sensitivity in tests using experimental fecal cloacal swab specimens that were spiked with ARV, AAV-1, IBDV, and CAV, and taken from specific pathogen free (SPF) chickens. This mPCR detected and differentiated various combinations of RNA/DNA templates from ARV, AAV-I, CAV, and IBDV without reduction of amplification from feces.

Animals↗

PubMed2002

Analysis of Dystrophin Gene Deletions by Multiplex PCR in Moroccan Patients.

Duchenne and Becker muscular dystrophy (DMD and BMD) are X-linked diseases resulting from a defect in the dystrophin gene located on Xp21. DMD is the most frequent neuromuscular disease in humans (1/3500 male newborn). Deletions in the dystrophin gene represent 65% of mutations in DMD/BMD patients. We have analyzed DNA from 72 Moroccan patients with DMD/BMD using the multiplex polymerase chain reaction (PCR) to screen for exon deletions within the dystrophin gene, and to estimate the frequency of these abnormalities. We found dystrophin gene deletions in 37 cases. Therefore the frequency in Moroccan DMD/BMD patients is about 51.3%. All deletions were clustered in the two known hot-spots regions, and in 81% of cases deletions were detected in the region from exon 43 to exon 52. These findings are comparable to those reported in other studies. It is important to note that in our population, we can first search for deletions of DMD gene in the most frequently deleted exons determined by this study. This may facilitate the molecular diagnosis of DMD and BMD in our country.

Journal Article↗

PubMed2000

Single-tube multiplex-PCR screen for common deletional determinants of alpha-thalassemia.

Alpha-thalassemia is very common throughout all tropical and subtropical regions of the world. In Southeast Asia and the Mediterranean regions, compound heterozygotes and homozygotes may have anemia that is mild to severe (hemoglobin [Hb] H disease) or lethal (Hb Bart's hydrops fetalis). We have developed a reliable, single-tube multiplex-polymerase chain reaction (PCR) assay for the 6 most frequently observed determinants of alpha-thalassemia. The assay allows simple, high throughput genetic screening for these common hematological disorders. (Blood. 2000;95:360-362)

Base Sequence↗

PubMed2003

Analysis of spontaneous, gamma ray- and ethylnitrosourea-induced hprt mutants in HL-60 cells with multiplex PCR.

AIM: To explore the molecular spectra and mechanism of human hypoxanthine guanine phosphoribosyl transferase (hprt) gene mutation induced by ethyluitrosourea (ENU) and (60)Co gamma-rays. METHODS: Independent human promyelocytic leukemia cells (HL-60) mutants at the hprt locus were isolated from untreated, ethyluitrosourea (ENU) and (60)Co gamma-ray-exposed cells, respectively, and verified by two-way screening. The genetic changes underlying the mutation were determined by multiplex polymerase chain reaction (PCR) amplification and electrophoresis technique. RESULTS: With dosage increased, survival rate of plated cell reduced (in the group with dosage of ENU with 100-200 micro g/ml, P<0.01; in the group with dosage of (60)Co gamma-ray with 2-4 Gy, P<0.05) and mutational frequency increased (in the group of ENU 12.5-200.0 micro g/ml, P<0.05; in the group of (60)Co gamma-ray with 1-4 Gy, P<0.05) significantly. In the 13 spontaneous mutants analyzed, 92.3 % of mutant clones did not show any change in number or size of exon, a single exon was lost in 7.7 %, and no evidence indicated total gene deletion occurred in nine hprt exons. However, deletions were found in 79.7 % of ENU-induced mutations (62.5-89.4 %, P<0.01) and in 61.7 % of gamma-ray-induced mutations (28.6-76.5 %, P<0.01). There were deletion mutations in all 9 exons of hprt gene and the most of induced mutations were chain deletion with multiplex exons (97.9 % in gamma-ray-induced mutants, 88.1 % in ENU-induced mutants). CONCLUSION: The spectra of spontaneous mutations differs completely from that induced by EUN or (60)Co gamma-ray. Although both ENU and gamma-ray can cause destruction of genetic structure, mechanism of mutagenesis between them may be different.

Alkylating Agents↗

PubMed1993

Fabry disease: detection of gene rearrangements in the human alpha-galactosidase A gene by multiplex PCR amplification.

Fabry disease, an X-linked recessive disorder of glycosphingolipid catabolism, results from lesions in the alpha-galactosidase A gene leading to deficient or absent activity of the lysosomal hydrolase. To facilitate the detection of rearrangements in this 14-kb gene, a method was developed for the PCR amplification of all seven exons from genomic DNA in a single multiplex reaction. The entire coding region and all the intron/exon boundaries were amplified as four products. Application of this method permitted the detection of all five partial deletions previously identified by Southern analysis. This rapid method can be used to identify gene rearrangements in affected hemizygotes and determine heterozygosity for at risk females in families with Fabry disease.

Base Sequence↗

PubMed1999

A multiplex-PCR/RFLP procedure for simultaneous CYP2E1, mEH and GSTM1 genotyping.

Inter-individual variation in metabolism of environmental toxicants, which is attributed to genetic polymorphism, may be a major risk factor in determining who will develop adverse health effects. This priority research area is the focus of many laboratories, and new techniques need to be developed to enhance the efficiency in generating data. We have developed and validated a new multiplex-polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) procedure for simultaneous genotyping of cytochrome P450 II E1 (CYP2E1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase mu (GSTM1). Enzymes from these three polymorphic genes are involved with the phase I and II metabolism of a variety of environmental toxicants. Therefore, simultaneous characterization of these genes will not only reduce costs but will increase the efficiency of data collection, thereby contributing to health risk assessment efforts.

Cytochrome P-450 CYP2E1↗

PubMed1995

Nonradioactive multiplex PCR screening strategy for the simultaneous detection of multiple low-density lipoprotein receptor gene mutations.

We have developed a rapid, nonradioactive screening test enabling the simultaneous analysis of three low-density lipoprotein receptor (LDLR) gene mutations (D154N, D206E, and V408M), which together account for familial hypercholesterolemia (FH) in approximately 90% of the South African Afrikaner population. The assay is designed so that FH patients, negative for these founder-related mutations (found in descendants of European settlers), subsequently can be screened for unknown mutations in the mutation-rich exon 4 of the LDLR gene. Our screening assay consists of two steps: (1) multiplex allele-specific PCR amplification of exons 4 and 9, and (2) simultaneous analysis of single- and double-strand conformational polymorphisms in exon 4 by vertical electrophoresis on low cross-linked polyacrylamide gels. The simplicity, specificity, and versatility of the multiplex assay makes it an ideal system for routine screening of FH mutations in large population samples.

Base Sequence↗

PubMed2000

Detection of two novel large deletions in SLC3A1 by semi-quantitative fluorescent multiplex PCR.

Cystinuria is an autosomal recessive aminoaciduria in which two clinical types have been described (type I and non-type I). Cystinuria type I is caused by mutations in SLC3A1, a gene located in 2p16 coding for an amino acid transporter named rBAT. Using multiplex semi-quantitative fluorescent PCR, we amplified the ten exons of SLC3A1 together with exon 5 of DSCR1 (located on chromosome 21) as a double-dose control gene. We detected two large novel deletions in a Belgian family, one comprising exons 2-10 and another one at exon 10. The method described here can be used to detect a range of deletions from single-base differences in size to entire missing exons, making it useful for scanning genes with a small to medium number of exons.

Amino Acid Transport Systems, Basic↗