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At least 685 records · Page 38Linked to original sources
PubMed2004

Validation of BIOMED-2 multiplex PCR tubes for detection of TCRB gene rearrangements in T-cell malignancies.

The BIOMED-2 Concerted Action BMH4-CT98-3936 on 'Polymerase chain reaction (PCR)-based clonality studies for early diagnosis of lymphoproliferative disorders' developed standardized PCR protocols for detection of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements, including TCR beta (TCRB). As no comparable TCRB PCR method pre-existed and only a limited number of samples was tested within the BIOMED-2 study, we initiated this study for further validation of the newly developed TCRB PCR approach by comparing PCR data with previously generated Southern blot (SB) data in a series of 66 immature (ALL) and 36 mature T-cell malignancies. In 91% of cases, concordant PCR and SB results were found. Discrepancies consisted of either failure to detect SB-detected TCRB rearrangements by PCR (6.5%) or detection of an additional non-SB defined rearrangement (2.5%). In 99% of cases (99/100), at least one clonal TCRB rearrangement was detected by PCR in the SB-positive cases. A predominance of complete Vbeta-Jbeta rearrangements was seen in TCRalphabeta(+) T-cell malignancies and CD3-negative T-ALL (100 and 90%, respectively), whereas in TCRgammadelta(+) T-ALL, more incomplete Dbeta-Jbeta TCRB rearrangements were detected (73%). Our results underline the reliability of this new TCRB PCR method and its strategic applicability in clonality diagnostics of lymphoproliferative disorders and MRD studies.

Blotting, Southern↗
PubMed2004

A simple and rapid genotyping method for beta-2 receptor (beta2 AR) gene using allele specific multiplex PCR.

BACKGROUND: Polymorphism of the beta2-adrenergic receptor (beta2 AR) gene is an important determinant of the function of this receptor. It affects receptor down-regulation and beta2-agonist responses. It has also been a focus of interest in attempts to elucidate the genetic basis of asthma, hypertension, obesity and cystic fibrosis. Several different techniques have been established to determine beta2 AR genotypes but none of these methods are simple enough to detect simultaneously all the five alleles of our research interest (Arg16/Gly16, -20T/C, Gln27/Glu27, -47T/C and Thr164/Ile164). OBJECTIVES: To develop a simple and rapid PCR based method for the simultaneous detection of five beta2 AR alleles. METHOD: DNA was extracted from whole blood using standard alkali lysis method. Primers specific at the 3' end for the polymorphic sites were designed. The nested allele specific PCR was optimized for reproducibility and specificity. Parameters investigated included concentrations of MgCl2, Taq polymerase, primers and annealing temperature, to produce specific bands of interest. DNA samples were selected at random and submitted for direct PCR sequencing. RESULT: The first PCR produced a fragment of size 710 bp, which was used as template for the subsequent duplex and triplex PCR to detect the mutation sites of the five alleles. The method was found reproducible and specific when used to genotype patients with bronchial asthma. The sequencing results confirmed the specificity of the PCR method. CONCLUSION: The simple and rapid method for the simultaneous detection of the five beta2 AR alleles is suitable for the study of beta2 polymorphism and its clinical consequences.

Alleles↗
PubMed2001

A single, multiplex PCR for differentiating all species of Trichinella.

The genus Trichinella is currently divided into seven species and at least three additional, unclassified genotypes, Trichinella T6, T8 and T9, where both T8 and T9 have been deemed very similar to T. britovi. Other than for the non-encapsulated species, the absence of distinguishing morphological characters and the overlapping nature of the biological characters within this genus make these traits unsuitable for diagnosis. Consequently, we have developed a simple PCR test for the unequivocal differentiation of all currently recognized species of Trichinella including Trichinella T6. DNA sequence data from each Trichinella genotype were generated from internal transcribed spacers, ITS1 and ITS2, and from expansion segment V (ESV) of the rDNA repeat, from which five different PCR primer sets were chosen. When used simultaneously, this primer mix generates a simple and unique electrophoretic DNA banding pattern for each species and genotype. The ESV-derived primer set contributes at least one band to each agarose gel-derived genotypic pattern and therefore functions as an internal control for PCR integrity. Geographical isolates of each Trichinella genotype were used to verify the reliability and reproducibility of respective DNA banding patterns using single muscle larvae.

Animals↗
PubMed2000

Subunit composition of rat ventral spinal cord GABA(A) receptors, assessed by single cell RT-multiplex PCR.

We analyzed the expression of native GABA(A) receptors in choline acetyltransferase and glutamic acid decarboxilase positive cells, from lamina IX of the lumbar region of rat spinal cord. More than one isoform of each subunit was detected within a single cell. The alpha3, alpha5, alpha1, beta3 and gamma2 subunit was the most frequent combination in both cell populations. However, the total number of subunit expressed by each cell type was different, being the ChAT positive cells the simplest. Interestingly, the ChAT and GAD positive cells also displayed a different pattern of distribution of both spliced isoforms of the gamma2 subunit. These results indicate that several GABA(A) receptors, with different molecular composition, are expressed in a single cell and that different cell types can express different GABA(A) receptors.

Animals↗
PubMed1994

Rapid detection of mycobacterial DNA in clinical samples by multiplex PCR.

Triplex-polymerase chain reaction technique (PCR) was developed for the detection and identification of mycobacterial DNA sequences in uncultured clinical samples. A 123 bp fragment corresponding to a specific Mycobacterium tuberculosis sequence complex, a 383 bp DNA fragment encoding for part of the 65 kD mycobacterial surface antigen, and a 268 bp fragment of the human beta-globin gene to demonstrate the presence of suitable DNA were amplified by triplex PCR. To demonstrate the applicability of this method, 206 alcohol-fixed, paraffin-embedded sputum samples from 47 patients with culture-proven tuberculosis were investigated. Of 206 samples, 157 were PCR positive, resulting in correct diagnosis of tuberculosis in 46 of 47 (97.8%) patients. Furthermore, 165 alcohol-fixed, auramin-stained sputum smears were examined in a blind trial. Triplex PCR revealed tuberculosis in 20 of 21 samples from patients with tuberculosis. In comparison, cultures were positive in 20 of 21 samples, and acid-fast organisms were found by microscopy in 18 of 21 samples. We conclude that triplex PCR is a rapid and sensitive technique for the detection of mycobacterial DNA in uncultured clinical samples and offers equivalent sensitivity (95.2%) and specificity (98.6%) as do culture methods.

Base Sequence↗