Multiplex polymerase chain reaction

About: Multiplex polymerase chain reaction is a research topic. Over the lifetime, 6409 publications have been published within this topic receiving 221244 citations.

Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936.

Abstract: Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

Abstract: This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

Detection of Potato mop top virus and Tobacco rattle virus Using a Multiplex Real-Time Fluorescent Reverse-Transcription Polymerase Chain Reaction Assay.

Abstract: Mumford, R. A., Walsh, K., Barker, I., and Boonham, N. 2000. Detection of Potato mop top virus and Tobacco rattle virus using a multiplex realtime fluorescent reverse-transcription polymerase chain reaction assay. Phytopathology 90:448-453. Tobacco rattle virus (TRV) and Potato mop top virus (PMTV) are important diseases of potato that are difficult to diagnose reliably by visual symptoms. Effective control strategies rely on accurate dia gnosis. This paper describes the development of a multiplex assay for the detection of TRV and PMTV directly from potato tubers and leaves by polymerase chain reaction (PCR) combined with in-tube fluorescent product detection (TaqMan). This technology obviates any post-PCR manipulations and has many advantages including reducing contamination risks, eliminating the need for ethidium bromide staining, and removing the time and cost of gel running. The new assay also allows the replacement of the two separate tests (a TRV reverse-transcription-PCR and a PMTV enzyme-linked immunosorbent assay) currently used with a single-tube multiplex format. In addition to greatly simplifying the detection of these two viruses, the multiplex TaqMan assay was also shown to be more sensitive than either of the tests that it replaces, allowing 100- and 10,000-fold increases in sensitivity for TRV and PMTV detection, respectively. The test reliably detected over 40 different isolates of TRV and PMTV obtained from a wide range of different cultivars and geographical locations, including some samples in which existing tests failed to detect virus. The use of an assay of this kind in routine diagnosis helps to speed up and streamline the diagnostic laboratory; in add ition, more reliable diagnosis should help in the control of this damaging disease.

Multiplexed Real-Time PCR Assay for Discrimination of Plasmodium Species with Improved Sensitivity for Mixed Infections

Abstract: The implementation of real-time PCR for the diagnosis of malaria has been hampered by poor sensitivity for the detection of mixed infections. We have optimized a method that enhances the sensitivity of detection of minor species in mixed infections within a single multiplex reaction. Our assay uses species-specific forward primers in combination with a conserved reverse primer and largely overcomes primer competition for the minor species DNA. With a blind panel of clinical samples, we successfully identified the species in 13/16 mixed infections. This assay was further validated with 91 blood samples and demonstrated a specificity and sensitivity for single infections of 100% compared with nested PCR as the "gold standard." This test has been implemented for routine confirmation of malaria species in Alberta, Canada. In comparison with species identification by microscopy, the real-time PCR test demonstrated greater sensitivity for the identification of species causing low-level and mixed infections and for the discrimination of Plasmodium species other than Plasmodium falciparum. Our experience supports a role for real-time PCR in the identification of malarial species in conjunction with microscopy.

Detection of amplified oncogenes by differential polymerase chain reaction.

Abstract: Oncogene amplification has been found in a variety of human cancers and may have prognostic importance. Therefore, techniques which facilitate detection of gene amplification could have wide applicability. We have devised a sensitive, rapid, and non-radioactive procedure for detecting alterations in gene copy number based on the polymerase chain reaction (PCR). In this technique, called differential PCR, a target gene and a single-copy reference gene are co-amplified by PCR in the same reaction vessel. The level of target gene amplification is reflected in the ratio between the two resulting PCR-product bands. We show that this method can detect as low as two-fold amplification of specific target genes. Furthermore, amplification of neu and the epidermal growth factor receptor gene could be detected in as few as 100 breast carcinoma cells or in single sections of formalin-fixed, embedded material.