Primer PCR Technique Research Articles

PCR assay a new approach for detection of enterotoxins

B. cereus group is considered as a potential problem, since it can contaminate many dairy products. In this study, three primer sets were selected to simultaneously detect two different species of the B. cereus group by using triple – primer PCR. The triple–primer PCR in this study were synthesized using the CER, CES and groEL genes for the detection of emetic toxin producing strains and another specific primer for the detection of diarrheal toxin (groEL gene only). Results indicated that all the diarrheal enterotoxin producing B. cereus strains showed a presence of groEL gene, while CER and CES genes were completely absent. Out of the six B. cereus strains tested for the production of diarrheal and emetic enterotoxins by using triple–primer PCR technique, three diarrheal enterotoxin producing strains were only detected. On the other hand, all six B. cereus strains had limited ability to produce emetic toxin. All diarrheal enterotoxin producing B. mycoides strains showed presence of groEL gene, but CER and CES genes were not detected in any of the B. mycoides tested strains. Consequently, four B. mycoides strains were tested for the production of diarrheal and emetic enterotoxins by using triple–primer PCR technique, only two strains showed diarrheal enterotoxin producing strains. In contrary all B. mycoides tested strains had limited ability to produce emetic toxin.

Molecular cloning, characterization, and function analysis of a mevalonate pyrophosphate decarboxylase gene from Ganoderma lucidum

This study investigated the role of the mevalonate pyrophosphate decarboxylase gene in the triterpene biosynthetic pathway of Ganoderma lucidum. The mevalonate pyrophosphate decarboxylase gene (mvd) was isolated using a degenerate primer-PCR technique. An analysis of the Gl-mvd transcription profile revealed a positive correlation between the expression of the Gl-mvd gene and triterpene content changes in G. lucidum during development. Furthermore, a promoter deletion analysis was conducted in G. lucidum to investigate the promoter activity and the role of methyl jasmonate (MeJA) responsive elements in the mvd promoter under the MeJA elicitor. The overexpression of Gl-mvd increased triterpene accumulation compared with the wild-type strain and increased the expression of several genes involved in the triterpene biosynthetic pathway. The findings of this study suggest that mvd may play an important role in triterpene biosynthesis regulation. Moreover, there may be the interactions among the genes involved in the triterpene biosynthetic pathway in the G. lucidum. Additionally, this study provides an approach for improving triterpene content through the overexpression of a key gene.

A fluorescence‐based sequence‐specific primer PCR for the screening of HLA‐B*57:01

Abacavir (ABC) is an antiretroviral drug highly effective in the treatment of HIV, but its intake can cause severe hypersensitivity reaction (HSR). A strong association between HLA-B(*)57:01 and ABC HSRs was reported by several studies, which demonstrated that HLA-B(*)57:01 screening had a 100% negative predictive value and that it could accurately identify patients at high risk of ABC HSRs. We propose a new sequence-specific primer PCR assay based on fluorescence detection through CE which is highly sensitive, allowing the use of non-infective sources of DNA such as saliva and buccal swabs, in addition to blood and reproducible, allowing automation of the analytical process. The results of our study were first compared with a standard sequence-specific primer PCR technique and reported a concordance of 100%, and then a blind external validation further confirmed the accuracy of our method.

Isolation of a fragment homologous to the rp49 constitutive gene of Drosophila in the Neotropical malaria vector Anopheles aquasalis (Diptera: Culicidae)

The constitutive ribosomal gene rp49 is frequently used as an endogenous control in Drosophila gene expression experiments. Using the degenerate primer PCR technique we have cloned a fragment homologous to this gene in Anopheles aquasalis Curry, a Neotropical vector of malaria. In addition, based on this first sequence, a new primer was designed, which allowed the isolation of fragments of rp49 in two other species, Aedes aegypti (Linnaeus) and Culex quinquefasciatus Say, suggesting that it could be used to clone fragments of this gene in a number of other mosquito species. Primers were also designed to specifically amplify rp49 cDNA fragments in An. aquasalis and Ae. aegypti, showing that rp49 could be used as a good constitutive control in gene expression studies of these and other vectorially important mosquito species.

Comparison of PCR-based mutation detection methods and application for identification of mouseSult1a1 mutant embryonic stem cell clones using pooled templates

Reverse genetic approaches to generate mutants of model species are useful tools to assess functions of unknown genes. Recent work has demonstrated the feasibility of such strategies in several organisms, exploiting the power of chemical mutagenesis to disrupt genes randomly throughout the genome. To increase the throughput of gene-driven mutant identification, efficient mutation screening protocols are needed. Given the availability of sequence information for large numbers of unknown genes in many species, mutation detection protocols are preferably based on PCR. Using a set of defined mutations in the Hprt1 gene of mouse embryonic stem (ES) cells, we have systematically compared several PCR-based point mutation and deletion detection methods available for their ability to identify lesions in pooled samples, which is a major criterion for an efficient large-scale mutation screening assay. Results indicate that point mutations are most effectively identified by heteroduplex cleavage using CEL I endonuclease. Small deletions can most effectively be detected employing the recently described "poison" primer PCR technique. Further, we employed the CEL I assay followed by conventional agarose gel electrophoresis analysis for screening a library of chemically mutagenized ES cell clones. This resulted in the isolation of several clones harboring mutations in the mouse Sult1a1 locus, demonstrating the high-throughput compatibility of this approach using simple and inexpensive laboratory equipment.