Multiplex polymerase chain reaction: A practical approach

Abstract

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.

A Sensitive, Specific, and Cost-Effective Multiplex Reverse Transcriptase-PCR Assay for the Detection of Seven Common Respiratory Viruses in Respiratory Samples

“…13 RT-PCR technology is not affected by these limitations because it is dependent on the presence of viral nucleic acid rather than infectious or intact virions. 18 The incorporation of an internal control in a PCR reaction is highly desirable, and we included human ERV to monitor inhibition of the PCR. However, our results showed that the majority of respiratory specimens do not contain inhibitory substances.…”

“…However, PCR may be affected by the presence of sequence variation that can be overcome by designing the assay to target highly conserved nucleic acid sequences. 18 Also, using conventional PCR technology to detect several viruses individually is labor-intensive and expensive. 18,19 These limitations can be overcome by using a multiplex PCR assay.…”

“…18 Also, using conventional PCR technology to detect several viruses individually is labor-intensive and expensive. 18,19 These limitations can be overcome by using a multiplex PCR assay. The multiplex format is a significant improvement over conventional PCR protocols, achieved by incorporating multiple primers that amplify RNA or DNA from several viruses simultaneously in a single reaction.…”

A highly sensitive and specific gel-based multiplex RT-PCR assay for the simultaneous and differential diagnosis of African swine fever and Classical swine fever in clinical samples

“…The most common problem is that quite a number of primers have to be used in the same reaction tube or well and these molecules may interact with each other, which may block the reaction [7,9,10,14,22]. A further problem may be the reliable identification of the various PCR products.…”

MegaPlex PCR: a strategy for multiplex amplification

“…Initial surface-based amplification cycles are then coupled to efficient solution-phase PCR using one common primer pair. We demonstrate this method by coamplifying and genotyping 75 unselected human single-nucleotide polymorphism (SNP) loci.The key challenge in developing a high-multiplex amplification procedure is preventing excessive off-target priming by the many primers in the reaction [1][2][3] . Several previously developed methods use surface immobilization of primer pairs as a way to separate reactions and thus limit primerdimer formation [4][5][6] , but drawbacks of these methods include inefficient amplification, loss of reactants from the surface and considerable primer-dimer formation within pairs of primers.…”