Multiplex PCR: Rapid DNA cycling in a conventional thermal cycler

Multiplex polymerase chain reaction (PCR) is a variant of PCR in which two or more target sequences are simultaneously amplified in the same reaction. In the present study we investigated the limits to which the duration of multiplex PCR steps can be shortened using the thermal cycler Gene Amp PCR system 9600 (Perkin Elmer, Oak Brook, IL). The present multiplex PCR assay simultaneously detects five different herpes viruses (HSV-1, HSV-2, VZV, CMV, and EBV) and assesses sample suitability in a single amplification round of 40 cycles. It appears that when six target sequences are simultaneously amplified in multiplex PCR, extension time is a critical parameter. Using a PCR protocol of 0 sec at 95°C, 0 sec at 60°C, and 0 sec at 74°C with Platinum Taq DNA polymerase (Life Technologies, Gaithersburg, MD), we were able to reduce the total cycling time of the multiplex PCR assay to as little as 55 min, without affecting the yield of PCR products or the specificity of the assay. It may be necessary to optimize each specific apparatus and template, but any such optimization would be trivial. © 2003 Wiley-Liss, Inc

Multicentre quality control of polymerase chain reaction for detection of HIV DNA.

International audienceOBJECTIVE: Seven French laboratories tested the specificity and sensitivity of the polymerase chain reaction (PCR) for the detection of HIV-1 DNA. METHODS: Following its own PCR protocols, each laboratory independently tested blind two panels of 20 coded peripheral blood mononuclear cell samples collected from HIV-1-seropositive individuals and from HIV-1-seronegative individuals at high or low risk of HIV infection. For the first panel, laboratories were free to select type and number of primers; for the second, all were required to use the two primer pairs Pol 3/4 and MMy 9/10' (Nef 1). RESULTS: False-positive and false-negative results were observed in all laboratories (concordance with serology ranged from 40 to 100%). In addition, the number of positive PCR results did not differ significantly between high- and low-risk seronegatives. The use of crude cell lysates in DNA preparation produced the same PCR results as phenol-extracted DNA. Discrepancies between laboratories indicated that factors other than primer pairs contributed strongly to laboratory variability. CONCLUSIONS: Our results emphasize the importance of both positive and negative controls in PCR and demonstrate the value of multicentre PCR quality control

Qualitative and quantitative detection of protein and genetic traits in genetically modified food

Due to the market introduction of genetically modified organisms (GMOs) in crops, foods, and ingredients, legislation worldwide came face to face with the question of the use and labeling requirements on GMO crops and their derivatives. In this review, protein- and DNA-based methods, such as enzyme-linked immunosorbent assay, western blots, and qualitative and quantitative polymerase chain reaction PCR (Q-PCR) are reviewed. Qualitative detection methods for genetically modified (GM) sequences in foods have evolved rapidly during the past years. The sensitivity of these systems is extremely high, even for processed foodstuffs. However, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. The recently introduced labeling threshold for GMOs in food ingredients by the European Union has forced official food control laboratories to apply quantitative PCR methods. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are discussed. As quantitative GMO detection methods measure GMO contents of samples in relation to reference material, priority must be given to international agreements and standardization on certified reference materials. The rapidly increasing number of GM foods on the market demands the development of more advanced multidetection systems, such as microarray technology. Challenges and problems arising from the inability to detect GM foods for which the modified sequence is unknown, the lengthy standardization procedures, and the need to continuously update databases comprising commercially available GM foods and the respective detection strategies are also discussed

Physical and immunological analysis of the two domains isolated from a variant surface glycoprotein of Trypanosoma brucei.

A specific surface glycoprotein of a variant of Trypanosoma brucei was cleaved with trypsin and the two major domains of the molecule have been purified. We have studied the chemical composition of each domain and compared the data to published results of the specific cDNA sequence. Circular dichroism measurements show that the amino-terminal domain includes preferentially alpha-helical or beta-sheet structure. The physicochemical analyses are supplemented by a prediction of secondary structure and a statistical pattern of hydrophilicity-hydrophobicity. The results are discussed in light of the internal limits that were described in the process of partial gene conversion occurring between the variant gene sequence and related members of the same gene family. Immunoblots with homologous antiserum indicate that the amino-terminal domain is implicated in antigenicity. In addition, immunoblotting with heterologous antiserum on native antigen, tryptic hydrolysates, or purified domains suggests a site of interaction supported by the two domains.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe