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Results for “Multiplex PCR”

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PubMed2006

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Chromosomes, Human, Pair 11↗

PubMed2005

Vi antigen expression in Salmonella enterica serovar Typhi clinical isolates from Pakistan.

The accurate identification of Salmonella enterica subsp. enterica serovar Typhi variants that fail to express the capsular polysaccharide, Vi, is an important and much discussed issue for medical microbiology. We have tested a multiplex PCR method which shows the presence or absence of the genetic locus required for Vi expression. Of 2,222 Salmonella serovar Typhi clinical isolates collected from patients' blood over a 4-year period in a region of Pakistan where typhoid is endemic, 12 tested negative for Vi expression by serological agglutination. However, only 1 of these 12 was Vi negative by the multiplex PCR method. This result was confirmed by immunofluorescence, the most sensitive method for Vi characterization in Salmonella serovar Typhi. The multiplex PCR described therefore represents a simple and accurate method for surveillance for Vi-negative variants of Salmonella serovar Typhi in Pakistan. Testing of clinical isolates of Salmonella serovar Typhi, before subculture, from other regions where Vi-negative Salmonella serovar Typhi has been described should be carried out so that the impact of vaccination with purified Vi antigen on the levels of Vi-negative Salmonella serovar Typhi in bacterial populations can be assessed.

Agglutination↗

PubMed2004

[Detection of micro mutation in dystrophin gene of DMD female carrier].

We attempted to identify a mutation in dystrophin gene in a female patient who was suspected a Duchenne muscular dystrophy (DMD) carrier with muscle weakness of upper limbs and congestive heart failure. We examined the mutation hot spots in DMD gene, exon 3, 6, 8, 13, 17, 19, 43, 44, 45, 47, 48, 49, 50, 52, 60 by multiplex PCR which had been a diagnostic screening strategy, and detected an extra band in exon 43 product. We also detected an extra band in exon 43 products by SSCP analysis for detection of small mutations which could not be detected by multiplex PCR. As a result of sequencing a PCR product of an exon 43, we confirmed an allele having the insertion of a 2 base of AT in Intron42, which is described for the first time. Although we can not conclude that this insertion is responsible for DMD, but it may cause abnormal splicing. In carrier detection of DMD without genetic information of proband, it is difficult to detect mutations by multiplex PCR solely. Therefore, SSCP of PCR products are recommended to detect mutation in DMD carrier.

Aged↗

PubMed2001

A multiplex PCR-based DNA assay for the detection of paraoxonase gene cluster polymorphisms.

Paraoxonase (PON) is a high-density lipoprotein (HDL) associated protein which is supposed to protect low-density lipoprotein (LDL) against oxidation and to play a role in the development of atherosclerosis. Interindividual variability in serum PON activity is attributable to common variants in components of the PON gene cluster on chromosome 7. We describe experimental conditions that permit the simultaneous determination of three common PON polymorphisms (PON1-192, PON1-55 and PON2-311) that are tightly associated with an increased risk of atherosclerosis. We used a multiplex PCR-based DNA assay using mismatch primers that introduce a unique recognition site for the endonuclease HinfI in the PCR products in case of presence of the R allele of PON 1-192, of the L allele of PON1-55 and of the S allele of PON2-311. The restriction analysis with HinfI allows to identify an electrophoretic band pattern which is specific for the combination of the three polymorphisms. This technique could be applied in the association studies aimed at assessing the role of PON and their polymorphisms in many clinical settings. In a preliminary study on a small population sample from south Italy about 10% of chromosomes exhibited the presumed risk-related haplotype R(192)/L(55)/S(311).

Alleles↗

PubMed2002

Rapid classification of partial waxy wheats using PCR-based markers.

Mutations in the three homeologous waxy loci Wx-A1, Wx-B1, and Wx-D1 of a waxy wheat line have previously been characterized at the molecular level. Using combinations of these mutations, six types of partial waxy wheat plus wild type and waxy wheat (types 1-8) can be produced. Here, we describe primer sets for all three loci that can be used under a single set of conditions, allowing 32 lines to be characterized as types 1-8 in a single PCR run using a 96-well plate. Using multiplex PCR, mutations at the Wx-B1 and Wx-D1 loci can be identified in a single PCR, reducing the number of reactions necessary to identify and select the desired partial waxy wheat line. A single multiplex PCR can be used to detect all three mutations when products are analyzed using capillary electrophoresis on a microchip device. The PCR conditions and primers are effective with a number of cultivars from other countries, indicating that the mutations found at the Wx-A1 and Wx-B1 loci of these cultivars likely have the same origins as the mutations in the corresponding loci of the waxy wheat line used in this study. The PCR selection method described here is an easy and effective alternative to the commonly used SDS-PAGE methods for identification of null alleles.

Amylose↗

PubMed2003

A multiplex polymerase chain reaction for a differential diagnosis of Plasmodium falciparum and Plasmodium vivax.

A multiplex PCR was designed for the differential diagnosis of the two parasite species by targeting the 18S rRNA gene with a set of primer combinations, amplifying DNA fragments of 1451-bp and 833-bp for P. falciparum and P. vivax, respectively. The sensitivity of this PCR test was high, as minimal as 0.1 parasite per one microliter of blood sample and a minimum of four copies of the target gene could be detected. For the diagnosis of mixed infection of two Plasmodium spp., there were no apparent competition or cross-reaction between the majority and minority Plasmodium species. The multiplex PCR was evaluated on 210 clinical samples and 60 normal controls. The PCR test yielded highly concordant results with microscopic examination, with the only one exception of a mixed (P. falciparum plus P. vivax) infection case, which was diagnosed as a single infection of P. falciparum by microscopy. We propose that the multiplex PCR is a sensitive, specific, and rapid tool that can serve as a useful differential diagnostic tool for detecting P. falciparum and P. vivax.

Animals↗

PubMed2001

Preimplantation genetic diagnostic protocols for alpha- and beta-thalassaemias using multiplex fluorescent PCR.

Haemoglobinopathies including alpha- and beta-thalassaemia are the world's most common class of single gene disorder. Prenatal diagnosis (PND) for beta-thalassaemia has been proven to be an effective strategy for controlling the incidence of new cases and is widely used in several countries where the disease is common. Successful preimplantation genetic diagnosis (PGD) protocols for beta-thalassaemia have been introduced using restriction fragment length polymorphism (RFLP), single-stranded conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE). However, contamination and allele dropout (ADO) remain an important concern for all of these strategies. In the present study two PGD protocols for detecting beta-thalassaemia mutations (codon 41-42 and IVSI-110) and one for alpha-thalassaemia (SEA mutation) have been designed and tested. These methods contain failsafe mechanisms to reduce the risk of misdiagnosis due to ADO or contamination and utilise multiplex fluorescent PCR (F-PCR). Interestingly, amplification efficiency and ADO were significantly affected by the choice of DNA polymerase and the freshness of the single cells used. The close similarity between the DNA sequences of beta-globin and delta-globin was also found to be an important issue that necessitated careful design of primers for the beta-globin gene.

Adult↗

PubMed1994

Detection of dystrophin gene deletion in Chinese Duchenne/Becker muscular dystrophy patients utilizing multiplex polymerase chain reaction.

Multiplex PCR was utilized to detect deletions at the hot spot regions of dystrophin gene. Thirty-four cases of DMD and 7 cases of BMD, based on their clinical manifestations, were examined. Eight normal male adults were included as control. Eleven DNA fragments coding the exons 1, 3, 6, 8, 13, 17, 43, 47, 50, 52 and 60 were chosen and primers were prepared with oligonucleotide synthesizer. Multiplex PCR was performed under the condition of 94 degrees C 1 minute for denaturation, 65 degrees C 3 minutes for annealing, 72 degrees C 3 minutes for extension. Thirty cycles were followed by a last cycle with 7 minutes extension. Clinically, there were no significant differences between Chinese patients and those reported in other areas except the low rate of positive family history (32%). The results from multiplex PCR showed that 15 cases (37%), 11 DMD and 4 BMD, proved to have deletions in the exons studied; 1 located at exons 6 and 8, 1 at exon 8, 1 at exons 8, 13 and 17, 1 at exons 13 and 17, 1 at exon 17, 3 at exon 43, 1 at exon 50, and 3 at exons 50 and 52, after false negatives were excluded. No difference in size or location was noticed between DMD and BMD in the sample-limited result.

Adult↗

PubMed2004

Multiplexed RT- PCR for high throughput screening applications.

To increase efficiency of high throughput gene expression profiling, we established a new TaqMan RT-PCR (real-time reverse transcriptase-polymerase chain reaction with internal probes for the quantification of PCR products) method for quantitative mRNA expression analysis. In this procedure, we utilized poly-A mRNA capture plates and validated a multiplexed single tube RT-PCR assay for cell culture applications, including compound testing via gene induction measurement. In the described procedure, all steps including RNA extraction, RT and PCR are performed in the same tube, thus significantly enhancing throughput of this method. Optimization of conditions, including the number of cells necessary for detection of mRNA signal was performed. With a relatively abundant message such as GAPDH, we saw a linear response for all of the concentrations tested, from 10,000 cells to 10 cells. We have also demonstrated multiplexing of different targets within the PCR reactions. In these experiments, we combined VIC-labeled probes for GAPDH with several FAM-labeled probes obtained from Assays On Demand (Applied Biosystems). In the reported experiments, multiplexing did not affect the efficiency of RT-PCR. We also demonstrated the utility of this technology for compound screening applications. The described technology also has the potential to accelerate studies on target and biomarker identification and toxicity assessment in ADMET (absorption, distribution, metabolism, elimination, and toxicity) testing.

Cell Count↗

PubMed2004

What is the best way to assess microsatellite instability status in colorectal cancer? Study on a population base of 462 colorectal cancers.

The assessment of the microsatellite instability (MSI) status in colorectal cancers is presently warranted for three reasons: 1) as a screening tool for hereditary nonpolyposis colorectal cancer, 2) as a prognostic marker, and 3) as a potential predictive factor of chemotherapy response. The aim of this study was to evaluate, on a large scale with tissue samples coming from a number of different sources, the difficulties met with routine use of immunohistochemistry (IHC) and to determine if it really does offer an accurate alternative to PCR genotyping. Colorectal carcinomas from 462 consecutive patients resected in public or private hospitals were assessed for MSI status by two methods: MSI testing (with BAT-26 microsatellite) and IHC detection of hMLH1, hMSH2, and hMSH6 proteins. Of the 398 cancers tested, immunohistochemistry was noncontributory in 42 (10.5%), focal in 9 (2.3%), and discordant with the PCR results in 36 (9%). For these 87 cases, complementary analyses were performed to explain discrepancy. After additional IHC assay with modified processing protocols, 8 cases remained noncontributory, 2 focal, and 28 discordant: 18 microsatellite stability IHC/MSI PCR and 10 MSI IHC/microsatellite stability PCR. For these discordant cases, we performed a multiplex PCR assay on DNA extracted from the frozen sample and BAT-26 was amplified from DNA extracted from the paraffin blocks used for IHC. Four discordant cases were reclassified after PCR multiplex assay (3 as MSI and 1 as microsatellite stability). Five other cases displayed intratumoral heterogeneity and 19 remained discordant. The discrepancy could be partly explained by variable technical protocols of fixation in the different laboratories, leading to variations in staining quality and difficulties in IHC interpretation. This population-based study is the first one to show that IHC is not sensitive and specific enough to be used routinely. Immunohistochemistry analysis of MMR proteins must be performed in standardized conditions and interpreted by confirmed pathologists. It cannot replace PCR as long as protocols are not optimized and harmonized.

Aged↗

PubMed2004

High prevalence of Helicobacter Species detected in laboratory mouse strains by multiplex PCR-denaturing gradient gel electrophoresis and pyrosequencing.

Rodent models have been developed to study the pathogenesis of diseases caused by Helicobacter pylori, as well as by other gastric and intestinal Helicobacter spp., but some murine enteric Helicobacter spp. cause hepatobiliary and intestinal tract diseases in specific inbred strains of laboratory mice. To identify these murine Helicobacter spp., we developed an assay based on PCR-denaturing gradient gel electrophoresis and pyrosequencing. Nine strains of mice, maintained in four conventional laboratory animal houses, were assessed for Helicobacter sp. carriage. Tissue samples from the liver, stomach, and small intestine, as well as feces and blood, were collected; and all specimens (n = 210) were screened by a Helicobacter genus-specific PCR. Positive samples were identified to the species level by multiplex denaturing gradient gel electrophoresis, pyrosequencing, and a H. ganmani-specific PCR assay. Histologic examination of 30 tissue samples from 18 animals was performed. All mice of eight of the nine strains tested were Helicobacter genus positive; H. bilis, H. hepaticus, H. typhlonius, H. ganmani, H. rodentium, and a Helicobacter sp. flexispira-like organism were identified. Helicobacter DNA was common in fecal (86%) and gastric tissue (55%) specimens, whereas samples of liver tissue (21%), small intestine tissue (17%), and blood (14%) were less commonly positive. Several mouse strains were colonized with more than one Helicobacter spp. Most tissue specimens analyzed showed no signs of inflammation; however, in one strain of mice, hepatitis was diagnosed in livers positive for H. hepaticus, and in another strain, gastric colonization by H. typhlonius was associated with gastritis. The diagnostic setup developed was efficient at identifying most murine Helicobacter spp.

Animals↗

PubMed2002

DNA genotyping of unbuffered formalin fixed paraffin embedded tissues.

Formalin-induced DNA degradation was studied at different fixation times (3, 7, 16 and 32 days) each on 10 formalin fixed paraffin embedded tissues (FFPET) stored for 15 years at room temperature. The four different extraction protocols used in this study showed that Chelex100 extracts performed the best at 3 and 7 days of formalin fixation (DFF) (with regard to the quantity and the quality of the DNA). However, Qiamp extracts showed better results for long sized alleles, as well for single polymerase chain reaction (PCR) amplifications after 16 and 32 DFF, as for multiplex PCR at shorter fixation times. DNA degradation is expressed by the size of the amplified alleles, only 100 bp templates surviving after 32 DFF (AMG locus). Single locus amplifications (CD4 and FES/FPS alleles) performed better than multiplex PCR (ProfilerPlus), with nearly 100% positive results at 7 DFF. In both types of amplifications, the success rate decreased proportionally with the time of formalin fixation and, consequently, with the size of the required DNA template.

Alleles↗

PubMed2000

Rapid detection of alpha-thalassaemia deletions and alpha-globin gene triplication by multiplex polymerase chain reactions.

We describe a sensitive, reliable and reproducible method, based on three multiplex PCR assays, for the rapid detection of seven common alpha-thalassaemia deletions and one alpha-globin gene triplication. The new assay detects the alpha0 deletions - -SEA, - (alpha)20.5, - -MED, - -FIL and - -THAI in the first multiplex PCR, the second multiplex detects the -alpha3.7 deletion and alphaalphaalphaanti3.7 variant, the third multiplex detects the -alpha4.2 deletion. This simple multiplex method should greatly facilitate the genetic screening and molecular diagnosis of these determinants in populations where alpha-thalassaemias are prevalent.

Gene Deletion↗

PubMed2001

Using MF-PCR to diagnose multiple defects from single cells: implications for PGD.

Single cell genetic analysis is generally performed using PCR and FISH. Until recently, FISH has been the method of choice. FISH however is expensive, has significant misdiagnosis rates, can result in interpretation difficulties and is labour intensive making it unsuitable for high throughput processing. Recently fluorescent PCR reliability has increased to levels at or surpassing FISH whilst maintaining low cost. However, PCR accuracy has been a concern due to allelic dropout. Multiplex PCR can now increase accuracy by using multiple markers for each chromosome to firstly provide diagnosis if markers fail and/or secondly confirm diagnosis. We compare a variety of diagnostic methods and demonstrate for the first time a multiplex PCR system providing simultaneous diagnosis and confirmation of the major aneuploidy chromosomes (21,18,13) and sex as well as DNA fingerprint in single cells. We also discuss the implications of using PCR for aneuploidy screening in preimplantation genetic diagnosis.

Aneuploidy↗

PubMed2005

Phenotypic and genotypic characterisation of Pasteurella multocida isolated from pigs at slaughter in New Zealand.

AIMS: To examine pigs at slaughter in New Zealand for the presence of Pasteurella multocida, and to determine for isolates, their biochemical profiles, somatic and capsular types, and the presence or absence of the HSB and toxA genes, associated with haemorrhagic septicaemia (HS) and progressive atrophic rhinitis (PAR), respectively. METHODS: Swabs from 173 lungs, 158 palatine tonsils and 82 nasal passages of pigs at two abattoirs in New Zealand were cultured for P. multocida using conventional techniques, and isolated colonies were subjected to biochemical tests for identification of biovars. Somatic serotyping was conducted using an agar gel immunodiffusion (AGID) test. Polymerase chain reaction (PCR) assays were used to confirm phenotypic identification of colonies using species-specific primers, capsule type using serogroup-specific primers and multiplex PCR, and to test for the presence of HSB and toxA genes. RESULTS: Pasteurella multocida was isolated from 11/173 (6.4%) lung, 32/158 (20.2%) palatine tonsil and 5/82 (6.1 %) nasal swab samples, a total of 48 isolates from 413 samples (11.6%). Isolation rates per farm ranged from 1-53% of tissue samples collected from pigs 5-6 months of age. On phenotypic characterisation, isolates were allocated to seven main biovars, viz 1, 2, 3, 5, 9, 12, and a dulcitol-negative variant of Biovar 8, the majority (30/48) being Biovar 3. Of the 42 isolates for which somatic serotyping was conducted, 10% were Serovar 1, 79% were Serovar 3, 2% were Serovar 6,1, 2% were Serovar 12, and 7% could not be typed. All 48 isolates were confirmed as P. multocida using a species-specific PCR. In the capsular multiplex PCR, 92% of isolates were Capsular (Cap) type A, 2% were Cap D, and 6% could not be typed. None of the samples were positive for the HSB or toxA genes. CONCLUSION: Serovars or capsular types of P. multocida associated with HS or PAR in pigs were not detected. Establishment of species-specific, capsular and toxin PCR assays allowed the rapid screening of isolates of P. multocida, while serotyping provided an additional tool for epidemiological and tracing purposes.

Abattoirs↗

PubMed2004

Measurement of relative copy number of CDKN2A/ARF and CDKN2B in bladder cancer by real-time quantitative PCR and multiplex ligation-dependent probe amplification.

Many tumors have large homozygous deletions of the CDKN2A locus (encoding p14(ARF) and p16) and of CDKN2B (p15). Our aim was to determine which gene is the major target in bladder cancer. We used quantitative real-time PCR (RTQ-PCR) to determine copy number of p15, of p14(ARF) exon 1beta, and p16 exon 2 in 22 tumor cell lines and 83 bladder tumors, some of which had been assessed previously by duplex PCR. Titration experiments showed that homozygous deletion could be detected in the presence of up to 30% normal DNA. Results for cell lines were compatible with previous cytogenetic analyses. Ten cell lines and 32 tumors (38.5%) had homozygous deletion of at least one target. Thirteen tumors (15.7%) had deletion of all three targets. Two tumors had deletion of p14(ARF) exon 1beta alone and four of p16 exon 2 alone. RTQ-PCR detected more homozygous deletions than duplex PCR. Finally we used a multiplex ligation-dependent probe amplification kit to provide independent confirmation of results. We conclude that with appropriate controls RTQ-PCR is a sensitive and robust method to detect copy number changes in tumors even in the presence of contaminating normal cell DNA.

Cell Cycle Proteins↗

PubMed1995

A simplified procedure for developing multiplex PCRs.

We have developed a simplified method for multiplex PCR based on the use of chimeric primers. Each primer contains a 3' region complementary to sequence-specific recognition sites and a 5' region made up of an unrelated 20-nucleotide sequence. Identical reaction conditions, cycling times, and annealing temperatures have been established for any PCR primer pair comprising the chimeric motif. Under these conditions, efficient multiplex amplification is achieved easily and reproducibly by simple adjustment of the individual primer concentrations. No additional modification of either the reaction components or annealing temperatures is required. The use of tagged primers provides a method for primer design that eliminates the multiple optimization steps involved in developing multiplex PCR.

Anemia, Sickle Cell↗

PubMed1995

Simultaneous detection of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by a rapid PCR method.

The identification of periodontal pathogens by conventional methods is time-consuming and difficult. Therefore, a multiplex PCR method for simultaneous detection of Actinobacillus actinomycetemcomitans (A.a.) and Porphyromonas gingivalis (P.g.) was developed for rapid and easy determination of these risk-indicator bacteria in human periodontal disease. The PCR primers were designed to hybridize to various regions of 16S rRNA genes, and a hot-start technique was used to obtain maximum sensitivity and specificity. This method can detect both of these bacteria in subgingival plaque samples at concentrations as low as 5 to 50 cells per sample. The sensitivity, however, was even 10 times better when the bacteria were analyzed in a water suspension. Since the only step between sample collection and the actual analysis is a brief centrifugation of the patient sample, the detection can be readily carried out in four hours. The performance of the method was studied with 36 patient samples. The results showed that the PCR method detected A.a. (44% vs. 25%, respectively) and P.g. (56% vs. 42%, respectively) more often than the conventional culture in plaque samples. Thus, our multiplex PCR method is rapid and more effective than conventional protocols in detecting these periodontal pathogens.

Aggregatibacter actinomycetemcomitans↗