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PubMed2003

[Application of multiplex polymerase chain reaction in rapid identification of Mycobacterium bovis BCG].

OBJECTIVE: To establish a method for the rapid identification of Mycobacterium bovis BCG. METHODS: A genomic region designated RD1 was found to be deleted from BCG strains, but present in other strains of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex (MTC) including Mycobacterium tuberculosis, Mycobacterium africanum, and Mycobacterium microti. With this information, a multiplex PCR method, developed to detect the deletion of RD1, was used to differentiate BCG strains from other strains of Mycobacterium bovis and other members of MTC. RESULTS: RD1 was shown to be absent in 5 BCG vaccine strains and 2 BCG strains isolated from an infant who died of systemic disseminated infection induced by BCG vaccination, but it was present in 3 Mycobacterium bovis standard strains, 6 Mycobacterium bovis strains isolated from diseased cows, deer or patients with pulmonary tuberculosis and other MTC strains including Mycobacterium tuberculosis H(37)Rv and H(37)Ra strains, 48 Mycobacterium tuberculosis strains isolated from patients with pulmonary tuberculosis, and 3 Mycobacterium africanum standard strains. CONCLUSION: The multiplex PCR method is simple, rapid, and specific for the identification of BCG among strains of MTC, and is applicable in clinical laboratories.

Mycobacterium bovis↗
PubMed2005

Evaluation of 2 real-time PCR assays for the investigation of mecA gene in clinical isolates of MRSA in Western Saudi Arabia.

OBJECTIVE: In diagnostic microbiology laboratories, Methicillin resistant Staphylococcus aureus (MRSA) is identified by positive coagulase test and positive deoxyribonuclease (DNase) activity followed by demonstration of oxacillin resistance on susceptibility testing on agar plate. This usually takes an approximately 48-72 hours. The purpose of this study is to evaluate 2 real-time polymerase chain reaction (PCR) assays for the presence of mecA gene in a population of MRSA strains circulating in Jeddah, Western Saudi Arabia, in order to determine their usefulness in the speedy diagnosis of MRSA in our clinical setting and their contribution to optimal patient management. METHODS: Ninety MRSA isolates obtained from clinical samples were identified by using conventional methods. They were collected between February 2004 and August 2004, from 2 major hospitals in Jeddah; King Abdul-Aziz University Hospital, Jeddah (50 strains) and King Khalid National Guard Hospital, Jeddah (40 strains). All isolates were confirmed as MRSA using Gram stain, catalase and coagulase activity, confirmatory DNAse activity and Kirby Bauer disc diffusion method with resistance to oxacillin by the agar disc method. The DNA extract was tested by 2 assays. The first was the commercial IVD Roche kit, which detects the mecA gene using the Light Cycler system. The other method employs multiplex PCR which detects As442 fragment and mecA optimized for the Smart Cycler system (Cephied). The length of time taken to perform the assays was recorded. RESULTS: All isolates were positive for Sa442 fragment and the coa gene specific for Staphylococcus aureus (S. aureus). However, 88/90 isolates (97.7%) tested were positive for mecA gene with both systems. The amplification, detection and melting curve analysis took 59.2 minutes for 32 samples on the Light Cycler and 46.7 minutes for 16 samples on the Smart Cycler. CONCLUSION: The 2 methods studied were equally specific and sensitive for the detection of mecA gene in confirmed S. aureus isolates and capable of identifying MRSA much earlier than conventional methods. The detection of 2 targets in the multiplex PCR assay reduces the 2-hour time required for DNase testing and may be used as a primary screening test for the detection of MRSA in clinical samples, such as blood cultures and sterile body fluids.

DNA Primers↗
PubMed2001

Analysis of microsatellite markers of Candida albicans used for rapid typing.

To obtain a rapid genotyping method of Candida albicans, three polymorphic microsatellite markers were investigated by multiplex PCR. The three loci, called CDC3, EF3, and HIS3, were chosen because they are on different chromosomes so as to improve the chances of finding polymorphisms. One set of primers was designed for each locus, and one primer of each set was dye-labeled to read PCR signals by using an automatic sequencer. Amplifications were performed directly from the colonies harvested on the agar plate without a sophisticated DNA extraction step. At total of 27 reference strains and 73 clinical independent isolates were tested. The numbers of allelic associations were 10, 22, and 25 for the loci CDC3, EF3, and HIS3, respectively. The combined discriminatory power of the three microsatellites markers was 0.97. The markers were stable after 25 subcultures, and the amplifications were specific for C. albicans. An initial study of 17 clinical isolate pairs, including blood culture and peripheral sites, showed a similar genotype for 15 of them, confirming that candidemia usually originates from the colonizing isolate. Therefore, microsatellite marker analysis with multiplex PCR and automated procedures has a high throughput and should be suitable for large epidemiologic studies of C. albicans.

Candida albicans↗
PubMed2005

Trichinella nativa in a black bear from Plymouth, New Hampshire.

A suspected case of trichinellosis was identified in a single patient by the New Hampshire Public Health Laboratories in Concord, NH. The patient was thought to have become infected by consumption of muscle larvae (ML) in undercooked meat from a black bear killed in Plymouth, NH in October 2003 and stored frozen at -20 degrees C fro 4 months. In January 2004, a 600 g sample of the meat was thawed at 4 degrees C, digested in hydrochloric acid and pepsin, and larvae were collected by sedimentation. Intact, coiled, and motile ML were recovered (366 larvae per gram (l pg) of tissue), which were passed into mice and pigs. Multiplex PCR revealed a single 127 bp amplicon, indicative of Trichinella nativa. The Reproductive Capacity Index (RCI) for the T. nativa-Plymouth isolate in mice was 24.3. Worm burdens in the diaphragms of two 3-month-old pigs given 2,500 ML were 0.05 and 0.2l pg by 35 days post-inoculation, while 2.2 and 0.75 l pg were recovered from two 3-month-old pigs given 10,000 ML; no larvae were recovered from four 1-year-old pigs given 2,500 ML (n=2) or 10,000 ML (n=2). Viable larvae were also recovered from frozen black bear meat harvested at two additional locations, one in southern Ontario, Canada, and one in upstate New York, USA. Multiplex PCR using genomic DNA from these parasite samples demonstrated that both isolates were T. nativa. This is the first report of the freeze-resistant species, T. nativa, within the continental United States.

Animals↗
PubMed2006

Prevalence of genes encoding exfoliative toxins among Staphylococcus hyicus isolated in Russia and Germany.

In the present study, previously characterized Staphylococcus hyicus isolated in Russia (n=23) and Germany (n=17) were investigated for the prevalence of the exfoliative toxin encoding genes exhA, exhB, exhC and exhD by multiplex PCR resulting in the detection of exhD positive strains among the S. hyicus isolated from pigs with exudative epidermitis in Russia and the detection of exhC and exhD for one and two strains isolated from exudative epidermitis in Germany respectively. The toxin gene negative strains were generally isolated from apparently healthy pigs, from other animals and from specimens where the relation between the isolation of S. hyicus and the clinical symptoms remained unclear. Partial sequencing of the toxin genes of selected exhC and exhD positive strains and comparing the sequencing results with sequences of exhC and exhD reference strains revealed an almost complete identity. The results of the present study were in agreement with the findings of Andresen and Ahrens (J. Appl. Microbiol., 96, 2004, 1265) and Andresen (J. Vet. Rec., 157, 2005, 376) that the presented multiplex PCR could be used to investigate S. hyicus for toxinogenic potential and that there is an association between the presence of toxin genes in S. hyicus strains from exudative epidermitis. However, comparable with the S. hyicus strains isolated in Germany which were investigated previously by Andresen (J. Vet. Rec., 157, 2005, 376), exhD seems to predominate in S. hyicus strains from Russia.

Amino Acid Sequence↗
PubMed1999

Detection of multiresistant Salmonella typhimurium DT104 using multiplex and fluorogenic PCR.

Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world. Salmonella typhimurium DT104 further poses a major health concern due to its acquisition of resistance to multiple antibiotics. The rapid detection of multiresistant S. typhimurium DT104 would facilitate strategies aimed at controlling this pathogen. We developed a specific and sensitive polymerase chain reaction (PCR) assay that amplifies a segment of DNA that is conserved in multiresistant S. typhimurium DT104. To provide further specificity for this PCR-based diagnostic test, we amplified two other gene fragments that are present in S. typhimurium DT104. A multiplex PCR containing primers for targeted sequences resulted in the amplification of predicted size fragments from S. typhimurium DT104 exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulphamethoxazole and tetracycline) or ASSuT resistance phenotypes. A minor modification of the multiplex PCR enabled the detection of other related multiresistant Salmonella such as S. typhimurium U302. To augment the detection process, we also designed a fluorogenic PCR assay that can detect the DNA of multiresistant S. typhimurium DT104 in the presence of excess contaminating bacterial DNA. These results provide a method by which multiresistant S. typhimurium DT104, or potentially the next emerging multiresistant Salmonella, can be accurately detected in only 3-4 h.

Drug Resistance, Microbial↗
PubMed1999

High-throughput genetic mapping in Arabidopsis thaliana.

To facilitate rapid determination of the chromosomal location of novel mutations, we have improved current approaches to gene mapping using microsatellite length polymorphisms. The high-throughput linkage analysis method described here allows a novel gene to be tested for linkage against the whole genome of a multicellular eukaryote, Arabidopsis thaliana, in a single polyacrylamide gel. The procedure is based on the simultaneous co-amplification of 21 microsatellites in a single tube, using a multiplex PCR mix containing 21 primer pairs, each including one oligonucleotide labeled with one of three fluorescent dyes that have different emission wavelengths. The amplification products, which range in number from 21 to 42, depending on the genotype of the individual being tested, are electrophoresed in a single lane on a polyacrylamide gel. The use of an automated fragment analyzer makes it possible to perform linkage analysis on a one gel-one gene basis using DNA samples from 19 F2 individuals obtained from an outcross involving a mutant and a wild-type that is genetically polymorphic with respect to the ecotype in which the mutant was generated. Discrimination of the amplification products is facilitated not only by labeling with different fluorochromes, but also by prior testing of different sequences for the ability to prime the amplification of each microsatellite, in order to ensure that multiplex PCR yields compatible amplification products of non-overlapping size. The method is particularly useful in large-scale mutagenesis projects, as well as for routine mapping of single mutants, since it reveals the map position of a gene less than 24 h after the F2 individuals to be analyzed have become available. The concepts employed here can easily be extended to other biological systems.

Arabidopsis↗
PubMed2002

MSH2 in contrast to MLH1 and MSH6 is frequently inactivated by exonic and promoter rearrangements in hereditary nonpolyposis colorectal cancer.

To estimate the relative frequency of mismatch repair genes, rearrangements in hereditary nonpolyposis colorectal cancer (HNPCC) families without detectable mutations in MSH2 or MLH1, we have analyzed by multiplex PCR of short fluorescent fragments MSH2, MLH1, and MSH6 in 61 families, either fulfilling Amsterdam criteria or including cases of multiple primary cancers belonging to the HNPCC spectrum. We detected 13 different genomic rearrangements of MSH2 in 14 families (23%), whereas we found no rearrangement of MLH1 and MSH6. Analysis of 31 other families, partially meeting Amsterdam criteria, revealed no additional rearrangement of MSH2. All of the MSH2 rearrangements, except one, corresponded to genomic deletions involving one or several exons. In 8 of 13 families with a MSH2 genomic deletion, the MSH2 promoter was also deleted, and the 5' breakpoint was located either within or upstream the MSH2 gene. This study demonstrates the heterogeneity of MSH2 exonic and promoter rearrangements and shows that, in HNPCC families without detectable MSH2 or MLH1 point mutation, one must consider the presence of MSH2 genomic rearrangements before the involvement of other mismatch repair genes. The simplicity and rapidity of their detection, using fluorescent multiplex PCR, led us to recommend to begin the molecular analysis in HNPCC by screening for MSH2 rearrangements.

Adaptor Proteins, Signal Transducing↗
PubMed2006

[Relationship of phenotype with type of deletion of dystrophin gene].

OBJECTIVE: To detect the distribution characteristics of dystrophin gene deletions in the northeastern of China and the relationship of severity with type of deletion. METHODS: To screen deletion distribution of 124 DMD/BMD patients via multiplex PCR, male high-risk fetuses were detected deletion by the same method. RESULTS: The deletion frequency was 49%. Deletions located in the regions of exons 45 - 53 and exons 8 - 19 were 41 (67%) and 13 (21%) cases respectively, and in 5 (8%) cases deletions were scattered over both regions, still 2 cases (3%) were checked up deletions lying in exons 34 and 43; there were 9 cases of in-frame deletions and 49 frameshift mutations in all deletions; of 30 high-risk fetuses 10 male ones were screened deletions, who had the same deletion-segments as their probands. CONCLUSIONS: The distribution of dystrophin gene deletions in the northeastern of China cluster mainly in two hot-spots, neighboring regions of exon 8 might be a real deletion "hot spot" in this region; the phenotype is associated with the type of gene deletion, the phenotype is BMD when in-frame deletions occur; severe DMD when frameshift mutations occur. Multiplex PCR method provides the short-cuts for detecting patients and making prenatal gene diagnosis.

Adolescent↗
PubMed2003

Multiplex polymerase chain reaction for the detection of herpes simplex virus, varicella-zoster virus and cytomegalovirus in ocular specimens.

PURPOSE: A majority of ocular viral diseases are caused by herpes group of viruses. Such infections, especially atypical herpetic keratitis, iridocyclitis and intra-ocular inflammations, can often present with overlapping clinical manifestations misleading the diagnosis. Molecular techniques are most useful in such instances for an accurate and rapid diagnosis since conventional methods are time consuming and less sensitive. A multiplex PCR was developed and used for the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) in ocular samples. METHODS: One hundred and forty six ocular samples (corneal scrapings - 52, aqueous fluid - 36, vitreous fluid - 31, tissues - 26, skin vesicle scraping - 1) were included in the study. The sensitivity of the assay was determined using serial dilutions of standard strains of HSV, VZV, and CMV vis-à-vis plaque forming assay. RESULTS: The sensitivity of the assay was 4, 4 and 12 PFU/ml or 20, 20 and 60 genome copy numbers of HSV, VZV and CMV respectively. Using DNA from various sources (fungal, bacterial, human leukocytes, tissues) along with standard positive controls, the assay was found to be highly specific. HSV DNA was detected in majority of the clinical samples (33.6%), most frequent being corneal samples. Comparatively, VZV and CMV infections were detected in small number of samples (VZV-3, CMV-2). CONCLUSIONS: We found the assay very useful in our set-up whenever a differential diagnosis of herpetic infections was suggested by the ophthalmologist. The multiplex PCR we have described here can be of greater value in clinics with larger number of patients suspected of having HSV, VZV or CMV infections.

Cytomegalovirus↗
PubMed2005

[Detection of integration status of human papillomavirus 16 in cervical precancerous lesions].

OBJECTIVE: To investigate the prevalence of integration of human papillomavirus 16 (HPV16) DNA into the host genome in cervical squamous intraepithelial lesions (SIL). METHODS: Multiplex PCR was used to detect the HPV/HPV16 infection and integration status of HPV16 in the surplus cells from liquid-based cytological samples from 108 patients with cervical cancer precursor lesions. Consensus primers GP5+/GP6+ were used to amplify a 150 bp long fragment in the conserved region of the HPV L1 gene so as to detect the presence pf HPV. Scion Image 4.0 electrophoresis image analysis soft was used to calculate the E2/E6 ratio so as to evaluate the episomal and integrated status of HPV16 infection: in episomal form, both targets should be equivalent, and in integrated form, E2 gene would be absent, while in mixed form of episomal/integrated mixed form, the copy number of E2 would be less than that of E6. RESULTS: Sixty-two out of the 108 patients (57.41%) had HPV infection. HPV16 were found in 32 of the 108 samples (29.63%). Among the 32 cases HPV16 DNA was exclusively episomal in 15 cases (46.88%), concomitant in 13 cases (40.62%), and integrated in 4 cases (12.50%). The prevalence of integrated and/or concomitant forms of HPV-16 DNA increased with progression of cervical disease. The prevalence of integrated form was 54.55% in the patients of CIN3 type, 50.00% in CIN2 type, 28.07% in CIN1 type, and 11.54% in the inflammatory type with significant differences between any 2 groups (all P < 0.01). CONCLUSIONS: HPV16 integration into the host genome is already present in some of CIN lesions. The multiplex PCR estimation of the HPV L1, HPV16 E2, E6 genes and E2/E6 ratio could be a simple method for detecting HPV/HPV16 infection and its integration status in liquid-based residual samples. It would be a helpful complementary tool for cytological screening to identify those patients at high risk of developing high-grade squamous intraepithelial lesions and cervical cancer.

Adult↗
PubMed2006

Usefulness of microchip electrophoresis for the analysis of mitochondrial DNA in forensic and ancient DNA studies.

We evaluate the usefulness of a commercially available microchip CE (MCE) device in different genetic identification studies performed with mitochondrial DNA (mtDNA) targets, including the haplotype analysis of HVR1 and HVR2 and the study of interspecies diversity of cytochrome b (Cyt b) and 16S ribosomal RNA (16S rRNA) mitochondrial genes in forensic and ancient DNA samples. The MCE commercial system tested in this study proved to be a fast and sensitive detection method of length heteroplasmy in cytosine stretches produced by 16 189T>C transitions in HVR1 and by 309.1 and 309.2 C-insertions in HVR2. Moreover, the quantitative analysis of PCR amplicons performed by LIF allowed normalizing the amplicon input in the sequencing reactions, improving the overall quality of sequence data. These quantitative data in combination with the quantification of genomic mtDNA by real-time PCR has been successfully used to evaluate the PCR efficiency and detection limit of full sequencing methods of different mtDNA targets. The quantification of amplicons also provided a method for the rapid evaluation of PCR efficiency of multiplex-PCR versus singleplex-PCR to amplify short HV1 amplicons (around 100 bp) from severely degraded ancient DNA samples. The combination of human-specific (Cyt b) and universal (16S rRNA) mtDNA primer sets in a single PCR reaction followed by MCE detection offers a very rapid and simple screening test to differentiate between human and nonhuman hair forensic samples. This method was also very efficient with degraded DNA templates from forensic hair and bone samples, because of its applicability to detect small amplicon sizes. Future possibilities of MCE in forensic DNA typing, including nuclear STRs and SNP profiling are suggested.

Animals↗
PubMed1998

Mutational scanning of mitochondrial DNA by two-dimensional electrophoresis.

An expedient, accurate, and cost-efficient test was developed to scan critical regions of the mitochondrial genome for all possible mutations by two-dimensional DNA electrophoresis. The test involves a two-step multiplex PCR amplification: a long-distance PCR to amplify almost the entire mitochondrial genome, which then serves as template for the amplification of 25 short PCR fragments in two multiplex groups corresponding to regions implicated in human diseases. The mixture of fragments was subsequently subjected to two-dimensional electrophoretic separation, first by size in a nondenaturant polyacrylamide gel and then on the basis of basepair sequence in a denaturing gradient polyacrylamide gel. This latter process of denaturing gradient gel electrophoresis is a most accurate form of mutation detection on the basis of differences in melting behavior of mutant and wildtype fragments. Evaluation of the method using samples with known homoplasmic and heteroplasmic mutations, as well as CEPH pedigrees to study segregation of polymorphic variants, indicated a very high accuracy; none of the previously identified mutations and polymorphisms escaped detection, and no erroneous segregation patterns of polymorphic variants were observed. In addition, two variants were found to be novel mutations when analyzed by sequence analysis. One of these novel mutations was a heteroplasmic mutation in the COXIII gene that was found to segregate to homoplasmy in the next generation. Heteroplasmic mutations as low as 1% of mtDNA could still be detected.

DNA Mutational Analysis↗
PubMed2001

Use of duplex polymerase chain reaction (duplex-PCR) technique to identify bovine and water buffalo milk used in making mozzarella cheese.

Molecular biology techniques have been used for species identification in food of animal origin in relatively recent years. A polymerase chain reaction (PCR) based method, the multiplex PCR, was recently applied to species identification in meat and meat products. It allows co-amplification of separate regions of a single gene or specific fragments, each typical of a different animal species in a single PCR reaction, using different pairs of primers in the same reaction mix. In the present paper, the duplex-PCR technique is proposed to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk). Because of its lower cost, undeclared bovine milk is added to water buffalo milk for making different kinds of mozzarella cheese. The results of this experiment indicate the applicability of this method, which showed an absolute specificity for the two species and a high sensitivity even down to low DNA concentrations (1 pg). In bovine and water buffalo mixtures of both milk and mozzarella cheese, the minimum concentration tested was 1% of bovine in water buffalo milk and water buffalo in bovine milk. The importance of the somatic cell content in raw milk is also discussed with special reference to the evaluation of mixtures (milk or cheese) of the two species.

Animals↗
PubMed2005

Multiplex PCR-single-base extension genotyping of multiple glutathione S-transferase polymorphisms.

Identification of genetic polymorphisms has recently gained increased interest, since they can be used as markers to identify the genes that predispose to disease. This emerging role of genetic polymorphism in clinical association has created the need for high-throughput genotyping methodologies. The present study describes the development of an SBE (single-base extension) methodology for the parallel identification of genetic polymorphisms in glutathione S-transferase genes, a superfamily of phase II drug-metabolizing enzymes. Oligonucleotide PCR primers were designed for simultaneous amplification of GSTM1, GSTP1 and GSTT1 gene loci SBE primers were also designed to be specific for each loci and to stop one nucleotide 5'-upstream of the polymorphic location. A specific tag was associated with each SBE primer to guarantee further discrimination by length. After simultaneous amplification of the target gene loci from genomic DNA extracted from human blood samples, SBE reactions were performed with fluorescently labelled dideoxynucleotide triphosphates. Individual genotypes were identified after separation of each tag-SBE probe by PAGE. The multiplex/SBE methodology was validated with previously genotyped DNA samples extracted from 21 individuals and it was used in a blind assay to genotype additional 64 individuals. The results show that SBE leads to the same results as the current 'gold standard' restriction-fragment-length-polymorphism-based genotyping methodologies, since SBE is a robust and accurate genotyping methodology that enables the parallel identification of multiple polymorphisms in the same reaction.

DNA Mutational Analysis↗