Multiplex Polymerase Chain Reaction MPCR

 

“MULTIPLEX POLYMERASE CHAIN REACTION”

Content: -

·        Introduction

·         Principle 

·         Advantages & Disadvantages

·         Types of Multiplex PCR

·         Optimization of Multiplex Reaction Components

·         NEB'S Tm Calculator

·         Primer Design parameters for Multiplex PCR

·         Advantages of Multiplex PCR

·         Application of Multiplex PCR

·         Some Multiplex PCR Kit Used in diagnosis 

·         Conclusion

·         References

Introduction 

In multiplex PCR, two or more primer sets designed for amplification of different targets are included in the same PCR reaction. Using this technique, more than one target sequence in a clinical specimen can be amplified in a single tube. As an extension to the practical use of PCR, this technique can save time and effort. The primers used in multiplex reactions must be selected carefully to have similar annealing temperatures and must be not complementary to each other. The amplicon sizes should be different enough to form distinct bands when visualized by gel electrophoresis. Multiplex PCR can be designed in either single-template PCR reaction that uses several sets of primers to amplify specific regions within a template, or multiple-template PCR reaction, which uses multiple, templates and several primers sets in the same reaction tube (Fig.). Although the use of multiplex PCR can reduce costs and time to simultaneously detect two, three, or more pathogens in a specimen, multiplex PCR is more complicated to develop and often is less sensitive than single-primer-set PCR. The advantage of multiplex PCR is that a set of primers can be used as internal control, so that we can eliminate the possibility of false positives or negatives. Furthermore, multiplex PCR can save costly polymerase and template in short supply.

     

                                                             Fig.- The overview of multiplex PCR.

Multiplex PCR represents a variant of PCR in which two or more DNA fragments are simultaneously amplified within a single reaction tube. This is achieved by including more than one primer pair to the reaction mixture. The approach is particularly relevant to food analysis, where it is often necessary to test for the presence of a variety of toxicants in a single sample. Multiplex reactions can usefully discriminate between real and false negative results. For this purpose, one set of primers is targeted at a target known to be present in the sample, while the second set targets the sequence of interest. The former allows the experimenter to distinguish between a true negative and a reaction failure. Multiplex primers must be designed so that each separate amplification product is of distinct size, to ensure that all fragments can be identified following amplicon separation by either agarose gel or capillary electrophoresis. Illustrating the simultaneous amplification of seven targets in a mixture of DNAs extracted from independent transgenic events in maize and soybean and non-transgenic maize and soybean. In this example, the primers were designed to amplify sequences within the transgenes and targeted in addition fragments of endogenous genes (zein in maize and lectin in soybean) to provide a negative control. Multiplex PCR – particularly when a significant number of primer pairs is involved – can require intricate optimization, and it may be in some instances be too difficult to achieve. Nevertheless, it represents an important technique for high-throughput analyses in a cost-effective manner.

·         Multiplex polymerase chain reaction (PCR), first reported in 1988, is a type of PCR in which two or more target sequences can be amplified simultaneously by including more than one pair of primers in the same reaction. 

·         Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. 

·         In a multiplexing assay, more than one target sequence can be amplified by using multiple primer pairs in a reaction mixture. 

·         As an extension to the practical use of PCR, this technique has the potential to produce considerable savings in time and effort within the laboratory without compromising on the utility of the experiment.

·         A multiplex PCR amplifies multiple targets within a single PCR run, in a single reaction vessel, from a single sample aliquot - instead of performing many separate reactions.

Principle

M-PCR is the simultaneous amplification of more than one target sequence in a single reaction tube using more than one primer pair. This co-amplification of two or more targets in a single reaction is dependent on the compatibility of the PCR primers used in the reaction. All primers in the reaction must have similar melting temperatures (Tm) so they anneal to and dissociate from complementary DNA sequences at approximately the same temperatures, allowing each amplification to proceed at the selected temperature. This procedure could not be done if one primer set was annealing at the time that another primer set was dissociating from its target. Therefore, all primers must be selected so their TmS are within a few degrees (°C) of each other. Each amplification proceeds independently of the others (as long as none of the reagents is present at rate-limiting concentrations) and each specific amplification product or amplicon is synthesized in an unencumbered way. Primers should also be chosen that define amplicons of approximately the same size range (100–500 bp), so each is synthesized efficiently and at equal rates. Each M-PCR assay must also have a detection step capable of identifying each amplicon. This can be done by gel electrophoresis with visual identification of separate amplicons of different size or by hybridization with specific DNA probes and detection using spectrophotometry, fluorometry, autoradiography, or chemiluminescence.

                                                      Fig.- Difference Between traditional PCR Vs Multiplex PCR

Multiplexing offers some important advantages that make it worth considering. When multiple target sequences are amplified in a single reaction, there is a substantial savings in master mix reagents. Half as many wells or fewer means half as much total dye, dNTPs, and more to acquire and spend – and that cost savings is appealing to many laboratories.

Additionally, pipetting issues sometimes mean that the total reaction volume isn’t the same between all qPCR reactions. This can be a problem when, for example, comparing a target gene to a reference gene analyzed in separate single plex reactions.

In a multiplex experiment, multiple genes are analyzed in a single reaction, so the volume between targets must be the same. With fewer wells to fill, setting up a multiplex experiment is also much faster than setting up an equal number of single plex reactions.

However, since qPCR should generally be run using identical triplicate reactions, even single plex experiments have a built-in way to monitor pipetting precision. For this reason, multiplexing is rarely, if ever, mandatory, but it can still represent a sizable savings in reagents and time if its complexities are managed.

                                                         Fig.- Identification of four different viruses from three patients

The extraction of genetic material from bacteria or viruses in a clinical specimen can provide template DNA for PCR. The amplification of a pathogen-specific DNA sequence can be interpreted as a ‘Positive’ result for an infectious disease. In recent years, multiplex PCR has expanded this simple paradigm.
The chances of appropriate treatment are increased if an infection is diagnosed correctly. However, many different infectious diseases can cause patients to present similar clinical symptoms, often resulting in misdiagnosis.

                                              Fig.- Identification of four different viruses from three patients

ADVANTAGES:

·         This technique has the potential to produce considerable savings in time and effort within the laboratory.

·         Without compromising on the utility of the experiment.

DISADVANTAGES:

·         Optimization is difficult; since many sets of forward and reverse primers are to be designed for use.

·         Increases cost.

·         Presence of multiple primer may lead to cross hybridization with each other and the possibility of miss-priming with other templates.

Types of Multiplex PCR 

Multiplexing reactions can be broadly divided in two categories:

1. Single Template PCR Reaction:-
This technique uses a single template which can be a genomic DNA along with several pairs of forward and reverse primers to amplify specific regions within a template.

2. Multiplex Template PCR Reaction:-
It uses multiple templates, and several primers sets in the same reaction tube. Presence of multiple primers may lead to cross hybridization with each other and the possibility of mis-priming with other templates.

         

                                                             Fig.- Multiplex PCR (Multiplex Templates) 

OPTIMIZATION OF MULTIPLEX REACTION COMPONENTS (REACTION MIX): 

·         Amount of Primer

·         dNTP and MgCl2, Concentrations

·         dNTP/MgCl2, Balance

·         PCR Buffer Concentration

·         Amount of Template DNA and Taq DNA Polymerase

·         Use of Adjuvants: DMSO, Glycerol, BSA

Reaction Mixture: 

Primers: -

·         Initially, equimolar primer concentrations of 0.1-0.5 µM each are used in the multiplex PCR.

·         When there is uneven amplification, with some of the products barely visible even after the reaction was optimized for the cycling conditions, changing the proportions of various primers in the reaction is required, with an increase in the number of primers for the "weak" loci and a decrease in the amount for the "strong" loci.

·         The final concentration of the primers (0.04-0.5 µM) may vary considerably among the loci.

dNTP and MgCl2 Concentrations: -

·         MgCl2 concentration is kept constant (2 mM),

·         While the dNTP concentration is increased stepwise from 0.5-1.6mM.

·         Optimization of Mg² is critical since Taq DNA polymerase is a magnesium- dependent enzyme.

PCR Buffer Concentration: -

·         Raising the buffer concentration to 2X improves the efficiency of the multiplex reaction but different concentrations of the buffer are optimised depending on the reaction mix.

Amount of Template DNA and Taq DNA Polymerase: -

·         The amount of template DNA is low (optimum), efficient and specific amplification can be obtained.

·         Different concentrations of Taq DNA polymerase are tested. (experimental optimization).

Use of Adjuvants: DMSO, Glycerol, BSA:

·         The most difficult multiplex PCR reactions can be significantly improved by using a PCR additive, such as DMSO, glycerol, BSA, which relax DNA, thus making template denaturation easier.

NEB'S Tm Calculator: -

                                         

                                                            Fig.- NEB’S Tm Calculator protocol

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·         Use the NEB Tm Calculator to estimate an appropriate annealing temperature when using NEB PCR products.

·         NEB offers the largest selection of recombinant and native enzymes for genomic research. While restriction enzymes remain part of our core product portfolio, our ever-expanding catalog also includes products related to PCR, gene expression, sample preparation for next generation sequencing, synthetic biology, glycobiology, epigenetics and RNA analysis. Additionally, we are also focused on strengthening alliances that enable new technologies to reach key market sectors, including molecular diagnostics development and nucleic acid vaccines.

 

Instructions

·         Select the product group of the polymerase or kit you plan to use.

·         Select the polymerase or kit from the list of products.

·         If needed, modify the recommended primer concentration.

·         Enter primer sequences (with up to 3 ambiguous bases). Spaces allowed.

Process followed for (5X) concentration: -

INITIAL DENATURATION: 95°C (1-2 min)

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DENATURATION: 95°C (5-30 sec)

                                               👇

ANNEALING: (30 sec -1 minute)

Optimized by doing a temperature gradient PCR, starting 5°C below calculated tm

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EXTENTION: Recommended: 68°C (1-2 minutes/kb)

Final extension: 68°C (5mins) Cycle number: 30-35 cycles give sufficient product.

PRIMER DESIGN PARAMETERS FOR MULTIPLEX PCR: -

Design of specific primer sets is essential for a successful multiplex reaction. The important primer design considerations described below are a key to specific amplification with high yield.

1. Primer Length

·         Multiplex PCR assays involve designing of large number of primers; hence it is required that the designed primer should be of appropriate length. Usually, primers of short length, in the range of 18-22 bases are used.

2. Melting Temperature

·         Primers with similar Tm. preferably between 55°C-60°C are used. For sequences with high GC content, primers with a higher Tm (preferably 75°C-80°C) are recommended. A Tm variation of between 3-5° C is acceptable for primers used in a pool.

3. Specificity

·         It is important to consider the specificity of designed primers to the target sequences, while preparing a multiplex assay, especially since competition exists when multiple target sequences are in a single reaction vessel.

4. Avoid Primer Dimer Formation

·         The designed primers should be checked for formation of primer dimers, with all the primers present in the reaction mixture. Dimerization leads to unspecific amplification.

·         All other parameters are like standard PCR primer design guidelines.

·         The most used dye for fluorescent- dye based detection is SyBr Green. The SyBr Green dye functions as an intercalating agent that emits detectable fluorescence when bound to double-stranded DNA.

ADVANTAGES OF MULTIPLEX PCR: -

1. Internal Controls

·         Potential problems in a simple PCR include false negatives due to reaction failure or false positives due to contamination. False negatives are often revealed in multiplex assays because each amplicon provides an internal control for the other amplified fragments.

2. Efficiency

·         The expense of reagents and preparation time is less in multiplex PCR than in systems where several tubes of Uniplex PCRs are used. A multiplex reaction is ideal for conserving costly polymerase and templates in short supply.

3. Indication of Template Quality

·         The quality of the template may be determined more effectively in multiplex than in a simple PCR reaction.

4. Indication of Template Quantity

·         The exponential amplification and internal standards of multiplex PCR can be used to assess the amount of a particular template in a sample. To quantitate templates accurately by multiplex PCR, the amount of reference template, the number of reaction cycles, and the minimum inhibition of the theoretical doubling of product for each cycle must be accounted.

APPLICATIONS OF MULTIPLEX PCR: -

Applications of multiplex PCR to the diagnosis of infectious diseases

During the past decade, advances in PCR technology and other DNA signal and target amplification techniques have resulted in these molecular diagnostics becoming key procedures. Such techniques are conceptually simple, highly specific, sensitive, and amenable to full automation. The most mature of these technologies, PCR, is in one variant or another now common in research laboratories and is used increasingly in routine diagnostic laboratory settings and undergraduate and high-school teaching. In diagnostic laboratories the use of PCR is limited by cost and sometimes the availability of adequate test sample volume. To overcome these shortcomings and to increase the diagnostic capacity of PCR, a variant termed multiplex PCR has been described. In multiplex PCR more than one target sequence can be amplified by including more than one pair of primers in the reaction. Multiplex PCR has the potential to produce considerable savings of time and effort within the laboratory without compromising test utility. Since its introduction, multiplex PCR has been successfully applied in many areas of nucleic acid diagnostics, including gene deletion analysis, mutation and polymorphism analysis, quantitative analysis, and RNA detection. In the field of infectious diseases, the technique has been shown to be a valuable method for identification of viruses, bacteria, fungi, and/or parasites. A representative list of such agents is shown in Table.

Based upon our own experience with multiplex PCR and those of other authors appearing in the literature during the last 10 years, we review the theoretical and practical basis of the development and optimization of multiplex PCR systems and discuss the application and potential of this technique in the field of diagnostic virology.

       Table1: - Representative list of applications of multiplex PCR to the diagnosis of infectious diseases

APPLICATION OF MULTIPLEX PCR IN DIAGNOSTIC VIROLOGY

During the last decade, several studies have demonstrated the practicality of identifying viral pathogens in many clinical and epidemiological settings using multiplex PCR (Table2). The technique has been used to screen for individual or symptom-associated viruses and examine associations of virus infection with disease. In addition, the technique has been shown to be a powerful and cost-effective tool for typing and subtyping virus strains in different epidemiological studies. PCR has proved to be a powerful tool for investigating meningitis and encephalitis caused by a variety of viruses. In neurological disease the requirement of rapid and reliable diagnosis to provide a rational basis for chemotherapy and limit unnecessary procedures and irrelevant therapy has driven development. The wide range of viruses associated with neurological disease includes herpes simplex virus (HSV); cytomegalovirus (CMV); varicella-zoster virus (VZV); Epstein-Barr virus (EBV); human herpes virus 6 (HHV-6); the enterovirus group, including echoviruses, polioviruses, and coxsackieviruses; adenoviruses; JC and BK viruses; arenaviruses; paramyxoviruses; rabies; and arboviruses. In view of the large number of potentially neuroinvasive viruses and because of the limited volume of the most useful diagnostic specimen—cerebrospinal fluid (CSF)—several multiplex PCRs have been developed

                            Table2.- Application of multiplex PCR for diagnosis of viral infections

Respiratory Viruses

 Viruses that commonly cause respiratory infection include respiratory syncytial virus (RSV), influenza viruses and parainfluenza viruses (PIV), and adenovirus, especially in infants and young children. Infection with these viruses may result in severe lower or upper respiratory tract disease requiring hospitalization. Thus, sensitive and rapid testing for these viruses is crucial to reduce the potential of nosocomial transmission to high-risk patients, limit unnecessary antibiotic use, and direct appropriate therapy following a specific diagnosis. For this reason, several studies have aimed to develop and evaluate multiplex PCR for detection of these viruses and provided substantial evidence of the utility of this technique as an important tool for management of patients presenting with respiratory infections. Several studies have utilized multiplex PCR to both detect and type or subtype influenza viruses, PIVs, and RSV in clinical specimens and are summarized below.

A nested multiplex RT-PCR which included three primer pairs in each round of amplification was utilized for the simultaneous detection, typing, and subtyping of influenza type A (H3N2 and H1N1) and type B viruses in a prospective surveillance of influenza in England in the 1995–1996 winter season. A total of 619 combined nose and throat swabs from patients with an influenza-like illness were analysed by culture and multiplex PCR. The multiplex RT-PCR detected influenza viruses in 246 (39.7%) samples compared to the 200 (32.3%) which yielded influenza viruses in culture. In addition, there was excellent correlation between the multiplex RT-PCR and culture for typing and subtyping of influenza viruses (100%) and for temporal detection of influenza A H3N2 and H1N1 viruses. It was concluded that whereas the multiplex RT-PCR demonstrated its utility in detection of influenza viruses in patients with influenza-like illness, patients with influenza-like illness who are negative for influenza viruses may harbor a pathogen(s) producing a syndrome difficult to distinguish clinically from true influenza (for example, RSV). Indeed, when this multiplex RT-PCR was modified so that it was capable of detecting and subtyping influenza A (H1N1 and H3N2) and B viruses as well as RSV subtypes A and B in respiratory clinical samples, the assay again demonstrated excellent (100%) correlation with the results of culture and serology. The ability of the test to detect viral coinfection in both simulated specimens and clinical samples was also demonstrated.

Ocular Infections

The benefits of PCR diagnostics over conventional techniques in the diagnosis of ocular infection are well documented for both the anterior and posterior segment of the eye. The development and evaluation of multiplex PCR for detection of adenovirus, HSV, and C. trachomatis in cases of keratoconjunctivitis demonstrated the feasibility of simultaneous screening for these agents. These studies further highlight the difficulties of multiplex PCR and provide substantial evidence for the importance of careful selection of oligonucleotide primers. A multiplex PCR was designed to detect adenovirus and HSV in eye swabs. The test produced results identical to those of virus isolation for 18 of 20 eye swabs (positive for adenovirus in five swabs, positive for HSV for five swabs and negative for adenovirus and HSV in eight swabs) but the remaining two specimens positive for adenovirus and HSV by virus isolation were negative by the multiplex PCR. However, the multiplex PCR proved superior to culture for the rapid diagnosis of viral keratoconjunctivitis. Replacement of the adenovirus primer pair to allow broader reactivity with adenovirus serotypes and inclusion of a primer pair targeting the cryptic plasmid of C. trachomatis yielded a triplex multiplex PCR. The sensitivity of this adenovirus-HSV-C. trachomatis multiplex PCR in comparison to a Uniplex PCR for the detection of adenovirus was 100%. However, the performance of the test to detect either HSV or C. trachomatis was relatively poor (69% compared to cell culture or 72% compared to an antigen detection technique). Selection of alternate HSV and C. trachomatis primer pairs allowed development of a multiplex PCR with identical sensitivity to that of Uniplex PCRs for detection of each of the three targets.

Other Applications

Several studies have utilized multiplex PCRs for detection and differentiation of human retroviruses. Four primer pairs were combined to detect the gag region of HIV type 1 (HIV-1), the env region of HIV-2, the pol region of human T-cell leukaemia virus type 1 (HTLV-1), and the tax region of HTLV-2. Amplicons were detected by liquid hybridization using 32P-end-labeled oligonucleotides. In the evaluation of a serologically well-established panel of singly and dually infected individuals, the assay detected 21 of 22 HIV-1, 8 of 10 HIV-2, 8 of 8 HTLV-1, and 8 of 8 HTLV-2 infections. The test was as sensitive as Uniplex PCRs and allowed the detection of coinfection. developed a multiplex PCR utilizing primer pairs targeting a portion of the gag region of HIV-1, the pol gene of HTLV-1 and -2, and a region of the HLA-DQ-α locus as an internal control. Products were analysed by automated capillary DNA chromatography (products can also be separated and visualized using gel electrophoresis and ethidium bromide staining). The test detected as few as 1 to 10 infected cells (2 to 20 target sequences) and was as sensitive as Uniplex PCRs.

Some Multiplex PCR Kit Used in diagnosis

QIAGEN Multiplex PCR Kit (100): -

For 100 x 50 µl multiplex PCR reactions: 2x QIAGEN Multiplex PCR Master Mix (providing a final concentration of 3 mM MgCl2, 3 x 0.85 ml), 5x Q-Solution (1 x 2.0 ml), RNase-Free Water (2 x 1.7 ml)

·         No optimization required, 

·         High specificity and sensitivity with a built-in hot start, 

·         Highly suited for many types of multiplex PCR applications, 

·         Easy to use and cost-effective

·         QIAGEN Multiplex PCR Kit (100)

·         QIAGEN Multiplex PCR Kit (1000)

          Fig.- QIAGEN Multiplex PCR Kit (100)

 

Platinum™ Multiplex PCR Master Mix: -

·         The Platinum™ Multiplex PCR Master Mix is designed specifically for endpoint multiplex PCR. Designed specifically for endpoint multiplex PCR, it supports easy multiplexing with minimal optimization.
 
• Multiplexing—Amplify up to 20 amplicons in a single reaction
• Flexibility—Amplify products from 50 bp to 2.5 kb
• Ease of Use—Perform multiplex PCR reactions with minimal optimization
 
A High-Specificity, High-Throughput Solution for Endpoint PCR
The performance of the Platinum™ Multiplex PCR Master Mix over a wide range of amplicon sizes permits the amplification of templates from 50 bp to 2.5 kb, greatly enhancing workflow flexibility. Coupled with its 20-plex capability and absence of primer dimers, it not only provides a high-throughput solution but also boasts high specificity through fewer non-specific primer binding events, and hence less reaction and primer waste

                Fig.- Platinum™ Multiplex PCR Master Mix

2X Multiplex Hot-Start PCR Master Mix: -

Features

·         Excellent performance and robustness in multiplex PCR

·         Optimized Master Mix for minimal hands-on and fast setup

·         Hot start for highest sensitivity and specificity

·         Exceptionally pure Hot Start Taq DNA Polymerase and highest quality dNTPs

Applications

·         Fast and high-throughput multiplex PCR

·         Parallel detection of multiple targets in a single assay

·         Gene expression analysis, diagnostic and forensic genotyping

·         Amplification of 50 bp to 2 kb targets

·         Purchase 2X Multiplex Hot-Start PCR Master Mix

·         Availability: In stock Bulk requests please inquire at oem@biotechrabbit.com

               Fig.- 2X Multiplex Hot-Start PCR Master Mix

 

Conclusion: -

Optimization of multiplex PCRs can prove difficult. A stepwise matrix-style approach may be followed; i.e., several optimal primer pairs are combined and the combination giving the best result is then chosen to be optimized or evaluated in a multiplex PCR format. Alterations of other PCR components over those usually described for most Uniplex PCRs have rarely improved the efficiency of the test. Recent developments in PCR technology, however, may facilitate the development of multiplex PCRs. The most appropriate of these seems to be the use of the nonmechanical hot start PCR.

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 quality control materials, and both external and internal quality controls must be rigorously applied. These must include the provision of both negative control specimens, and, for each nucleic acid target, a positive control designed to ensure early signalling of any reduction in test sensitivity from assay to assay. 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. In addition, attention should be given to primer pairs detecting multiple strains or types to ensure the identification of as many strains of the target species as possible. Where possible, robust (i.e., reliable in routine diagnostic settings) multiplex PCRs that do not use nested primers are preferable to avoid contamination to rationalize use in routine settings, and to facilitate automation.

References: -

1)      Elnardo EM, Ashshi AM, Cooper RJ, Klapper PE. Multiplex PCR: optimization and application in diagnostic virology. Clinical microbiology reviews. 2000 Oct 1;13(4):559-70.

2)      Edwards MC, Gibbs RA. Multiplex PCR: advantages, development, and applications. Genome Research. 1994 Feb 1:3(4):S65-75.

3)      Markoulatos P, Siafakas N, MoncanyM. Multiplex polymerase chain reaction: a practical approach. Journal of clinical laboratory analysis. 2002 Jan 1;16(1): 47-51.

4)      Henegariu O, Heerema NA. Dlouhy SR. Vance GH, Vogt PH. Multiplex PCR: critical parameters and step-by-step protocol. Biotechniques. 1997 Sep 1:23(3): 504-11.

5)      Krause JC, Panning M. Hengel H. Henneke P. The role of multiplex PCR in respiratory tract infections in children. Dtsch Arztebl Int. 2014 Sep 19:111(38): 639-45.