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M Moncany

Publications and source records attributed to M Moncany.

5 recordsLinked to original sources
PubMed2003

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 degrees C, 0 sec at 60 degrees C, and 0 sec at 74 degrees 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.

DNA↗
PubMed2002

Multiplex polymerase chain reaction: a practical approach.

Considerable time and effort can be saved by simultaneously amplifying multiple sequences in a single reaction, a process referred to as multiplex polymerase chain reaction (PCR). Multiplex PCR requires that primers lead to amplification of unique regions of DNA, both in individual pairs and in combinations of many primers, under a single set of reaction conditions. In addition, methods must be available for the analysis of each individual amplification product from the mixture of all the products. Multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. The development of an efficient multiplex PCR usually requires strategic planning and multiple attempts to optimize reaction conditions. For a successful multiplex PCR assay, the relative concentration of the primers, concentration of the PCR buffer, balance between the magnesium chloride and deoxynucleotide concentrations, cycling temperatures, and amount of template DNA and Taq DNA polymerase are important. An optimal combination of annealing temperature and buffer concentration is essential in multiplex PCR to obtain highly specific amplification products. Magnesium chloride concentration needs only to be proportional to the amount of dNTP, while adjusting primer concentration for each target sequence is also essential. The list of various factors that can influence the reaction is by no means complete. Optimization of the parameters discussed in the present review should provide a practical approach toward resolving the common problems encountered in multiplex PCR (such as spurious amplification products, uneven or no amplification of some target sequences, and difficulties in reproducing some results). Thorough evaluation and validation of new multiplex PCR procedures is essential. The sensitivity and specificity must be thoroughly evaluated using standardized purified nucleic acids. Where available, full use should be made of external and internal quality controls, which must be rigorously applied. As the number of microbial agents detectable by PCR increases, it will become highly desirable for practical purposes to achieve simultaneous detection of multiple agents that cause similar or identical clinical syndromes and/or share similar epidemiological features.

Humans↗
PubMed1992

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

OBJECTIVE: 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.

Base Sequence↗
PubMed1990

Polymerase chain reaction for studies of mother to child transmission of HIV1 in Africa.

The feasibility and implications of the use of the polymerase chain reaction (PCR) assay in studies of HIV1 mother to child transmission in Africa were investigated. Uncultured leukocyte blood cells (PBL) obtained in Brazzaville (Congo) from newborns and infants (mean age = 27 weeks) of infected mothers were tested. HIV1 DNA sequences were identified in the PBL of six of eight newborns and 14 of 23 babies born to HIV1-positive mothers. In addition two of four babies, who at birth had been seropositive and subsequently were seronegative, were HIV1 DNA positive by PCR. This study demonstrates directly, therefore, a high rate of HIV1 transmission in Africa; it also indicates that PCR should be used for such epidemiological studies.

Africa↗