multiplex PCR
summary
Multiplex polymerase chain reaction (MPCR), also known as compound PCR, is a new PCR amplification technique improved and developed on the basis of conventional PCR, i.e., more than two pairs of primers can be added to a reaction system to amplify multiple nucleic acid fragments simultaneously, which was first proposed by Chambehian in 1988. It was first introduced by Chambehian in 1988, and its reaction principle, reagents and procedure are the same as those of conventional PCR. Multiplex PCR has the specificity and sensitivity of a single PCR, but is also faster and more economical, showing great flexibility in the design of primers and PCR reaction conditions. Multiplex PCR also provides an internal control that indicates the relative quantity and quality of template.
principle
The basic principle of multiplex PCR is the same as that of conventional PCR, with the difference that more than one pair of primers is added to the same reaction system. If there are templates complementary to each pair of primers, they will bind to the corresponding parts of the templates, and more than one fragment of the target DNA will be amplified in the same reaction system at the same time. The composition of the multiplex PCR reaction system and the conditions of the PCR cycle need to be optimized to ensure that several fragments are amplified at the same time. “Theoretically, as long as the conditions for PCR amplification are appropriate, there can be an unlimited number of primer pairs, but in practice, the number of pairs that can be amplified is limited due to a variety of constraints.
PCR results can be analyzed by several methods, including agarose gel electrophoresis, polyacrylamide gel electrophoresis, nucleic acid hybridization, restriction enzyme digestion, and nucleic acid sequencing. Agarose gel electrophoresis is the simplest and fastest method, but it has a lower resolution compared with polyacrylamide gel electrophoresis. Restriction enzyme digestion requires the recovery of PCR products by digestion and electrophoresis, which is time-consuming and difficult to apply. Nucleic acid sequencing is the most reliable method for identifying PCR products, but it requires specialized sequencing equipment and is time-consuming. Nucleic acid sequencing is the most reliable method for identifying PCR products, but it requires specialized sequencing equipment and is time-consuming. Agarose gel electrophoresis is the preferred multiplex PCR method for identifying infectious disease pathogens for use in the clinic and for epidemiological investigations in the field.
Applications
Common applications of multiplex PCR are listed below:
(I) Application of multiplex PCR in genetic disease diagnosis
1. Detection of DMD and BMD
Duchenne muscular dystrophy (DMD) is a very common human genetic disease, which is an X-chromosome recessive hereditary muscle degeneration disease. 50% of cases are caused by gene deletion, and the disease occurs in about one out of every 3,500 male infants, and 1/3 of cases are caused by new mutations. The myotonic dystrophy gene is 2000kb long, with at least 70 exons spaced by 35 introns averaging 35kb in length. There are no effective treatments available, so highly accurate prenatal diagnosis and screening for DMD positivity is essential. In the past, Southern analysis was used for diagnosis, but because the gene consists of up to 70 exons, at least 7-9 cDNA clones are required for diagnosis, which is expensive, time-consuming, and difficult to be performed routinely.BMD stands for Becker muscular dystrophy, which is also known as an X-chromosome recessive inherited muscle degeneration disease. The incidence of BMD is 1 in 30,000 newborn boys. 65% of BMD cases are due to a genetic deletion.
Chamberlain et al. detected DMD by multiplex PCR and designed six pairs of exon primers. The primers were used in 1 μmol/L with 10U Taq enzyme, and the extension temperature was 72C for 3 min and 25 cycles. After electrophoresis, the products were considered to be abnormal if the bands were missing or shifted when compared with the corresponding bands of the normal control. Subsequently, the laboratory increased the number of primer pairs to nine, which allowed the identification of at least 80% of DMD gene deletions and the direct diagnosis of more than 50% of cases. Hentemann et al. designed two pairs of primers with products of 140bp and 73bp, respectively, which were used to test 42 patients, and seven deletions were identified and confirmed by Southern hybridization. Simard et al. reported the amplification of DMD with Chamberlain's primers and improved the DNA template processing by direct PCR amplification of cells after centrifugation with 1 ml of amniotic membrane solution and lysis with non-ionic detergent and PCR buffer with proteinase K. The results were satisfactory. Since DMD/BMD is an allele, the recent literature has focused on the simultaneous detection of these two diseases. Beggs et al. used multiplex PCR to simultaneously detect the eight exons and promoter of the myotonic dystrophy gene, which led to the diagnosis of 98% of DMD/BMD with deletion of the gene. Covone et al. used two methods to control 127 cases of DMD/BMD: one method was to use nine cDNA probes related to DMD to hybridize with gene family DNA digested by HindJH, and the other method was to use nine pairs of primers for DMD exons to conduct multiplex PCR, and the results were 73 cases (57%) with gene deletion, and the results of the two groups of methods were similar. Scholars from West China Medical University also tested 17 DMD/BMD cases with Chamberlain's 9 pairs of primers, and found deletions in 8 cases (47%), which proved that primer sequences targeting Westerners can also detect Chinese cases.
2. Detection of other genetic diseases
Picci et al. screened for mutations in cystic fibrosis (CF) by multiplex PCR with four exon pairs of primers, followed by digestion of the PCR products with restriction endonucleases and vertical polyacrylamide gel swimming. 15 cases were examined, and three were found to have mutations. 8 pairs of primers were designed to test for DMD/BMD and CF, and the DMD/BMD deletion mutation was screened by gel electrophoresis. Prior et al. designed eight pairs of primers to detect DMD/BMD and CF simultaneously, screened for DMD/BMD deletion mutations by gel electrophoresis, and determined the presence of CF mutations by allele-specific oligonucleotide hybridization. It was shown that sufficient DNA could be obtained from blood spots by multiplex PCR for molecular analysis and could be used for neonatal screening. Pillers et al. studied a case of concurrent AIED (Aland island eye disease) glycerol kinase deficiency (GKD) and DMD by multiplex PCR and Southern hybridization, and found that DXS67 (l-deoxyribonucleic acid) was the most common mutation in DMD/BMD. A deletion between the DXS67 (l-deoxy-D-xylulose 5-phosphate synthase 67) and DMD genes was found. Multiplex PCR has also been reported to screen for steroid sulfatase (STS) deficiency and to diagnose patients with thalassemia on the day of diagnosis.
spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder that causes lower motor neurons in the base of the brain and spinal cord to split, preventing them from sending the chemical and electrical signals that muscles need to perform their normal activities. sMA primarily affects the proximal muscles, which are the closest to the trunk. SMA primarily affects the patient's proximal muscles, the muscles closest to the trunk. Non-random muscles that control the movement of organs such as the stomach, intestines, and bladder are not affected. All types affect nerve cells called motor neurons, which control the movement of random muscles. Recent genetic studies have found that motor neurons may die because one or more proteins are missing, or because they are unable to perform their full function. simard et al. applied multiplex real-time reverse transcriptase-PCR to the survival motor neuron (SMN), which is the most common type of neuron in the world. Simard et al. applied multiplex real-time reverse transcriptase-PCR to quantify the transcripts of survival motor neuron (SMN) in blood samples from 42 patients with latent SMA, and three time points were taken from each patient. They found that the expression of SMN was stable at different time points of each patient, and the real-time quantification of SMN mRNA expression can be used as a biomarker for the clinical trials of SMA.
Mannose-binding lectin (MBL) is a serum protein that triggers complement activation and thus plays an important role in innate immunity. Low levels of MBL impair the regulatory role of pathogenic microorganisms, leading to recurrent infections in children, severe infections in immunocompromised patients, and autoimmune diseases.The gene encoding MBL is located on human chromosome 10q11.2 to q21 and consists of four exons, and serum levels of MBL are affected by promoter polymorphisms and mutations in the first exon of the gene. Currently, the following methods are used for MBL genotyping: PCR-restriction enzyme digestion, sequence_specific oligonucleotide probes (SSOP), amplification refractory mutation system (ARMS) and the MBL genotype. mutation system (ARMS), ARMS combined with SSOP, and hetero duplex analysis.) Real-time PCR and 5'-end nuclease analysis using a groove-conjugated DNA probe. Helena et al. developed a rapid and efficient multiplex PCR method for typing the MBL2 gene and applied it to study the allele frequency of MBL2 in a representative sample of Slavs from the Czech Republic. A total of 359 unrelated Czech MBL2 genes were analyzed, and it was concluded that the LYD haplotype was more common in Slavic ancestry than in other Caucasian populations, which also proved that multiplex PCR is a faster, simpler, and more time- and labor-saving method for MBL2 typing than traditional PCR.
According to current research in the field of infertility, about 10-15% of couples have infertility problems, and male infertility accounts for about 50% of all infertile individuals, of which 40-50% have sperm defects such as oligospermia and azoospermia. The long arm of the human Y chromosome is required for sperm production. Deletions in three different regions result in severe sperm defects, including non-obstructive azoospermia and hypospermia. These regions are known as the azoospermia factor (AZF), and the three separate non-overlapping regions, defined as AZFa, AZFb, and AZFc, are associated with the production of damaged sperm in humans. Recent studies have demonstrated that microdeletions in the Yq chromosome are transmitted to their sons. Yeom et al. used multiplex PCR to amplify six loci, including the sex-determining region on the Y chromosome (SRY) as a positive control, and five sequence-tagged sites (STS) in the region of the spermless spermatid factor as a positive control. The primers in this study were Cy3-labeled, and the PCR products could be hybridized to fixed probes, providing a sensitive, high-throughput method for the detection of Y-chromosome deletions and a new approach to screening for male infertility.
(二) Preimplantation genetic diagnosis (PGD)
PGD was developed more than 10 years ago with the primary goal of identifying genetic disorders caused by mutations or chromosomal errors. It was first used to detect X-linked disorders in couples, and then applied to different patients to achieve eugenics, including carriers of single-gene disorders, whether dominant or recessive, autosomal or X-linked; carriers of chromosomal structural anomalies, including translocations, flips, deletions, insertions, etc.; avoiding chromosomal anomalies in offspring of high-risk mothers; couples with repeated implantation failures of assisted reproduction treatments; and couples with repeated unexplained miscarriages. Couples with repeated unexplained miscarriages. Currently, PGD has become an alternative to prenatal diagnosis.
For patients with single gene defects, multiple loci can be amplified simultaneously by multiplex PCR. Selecting polymorphic marker loci located on the same chromosome or close to the causative gene can effectively diagnose mutant loci and polymorphic alleles, analyzing multiple diagnostic loci at the same time and reducing the probability of misdiagnosis. In addition, highly variable fingerprinting sites can be amplified to indicate whether the DNA template is contaminated.
Cystic fibrosis (CF) is the first successful application of single-cell preimplantation genetic diagnosis of a single gene defective disease.The mutational spectrum of CF varies widely, and therefore, the development of a mutation-specific PGD method is not feasible.1 In the literature, a universal multiplex PCR method for CF has been established for embryonic genetic diagnosis. A universal multiplex PCR method for CF has been developed in the literature for the genetic diagnosis of embryos. In this study, four closely interlinked highly polymorphic repeat identifiers on both sides of the CF transmembrane regulator (CFTR), D7S523, D7S486, D7S480 and D7S490, were used. 100 leukocytes and 50 oocytes were examined, 99% of which yielded multiplex PCR results. A total of 100 leukocytes and 50 oocytes were tested, of which 99% yielded multiplex PCR results, and the overall allelic drop out (ADO) frequency ranged from 2% to 5%. After confirming the presence of ADO and additional alleles, 95% of the multiplex PCR results could be used to establish genotypic markers. Based on parental genotypes, approximately 90% of embryos can be reliably genotyped for PGD using a single cleavage sphere, taking into account the loss of embryo transmission due to variant parameters (5%), ADOs (0-2%), and single recombination (1.1%-3%). The probability of misdiagnosis is comparable to the known probability of double recombination for both markers, which is less than 0.05%. Thus, this polymorphism and multi-allele labeling system is a reliable alternative to direct genetic diagnosis of mutant embryos.
(三) Application to genetically modified organisms (GMO)
In recent years, there have been reports on the application of multiplex PCR technology in the qualitative and quantitative detection of GMOs. Chen Wenbing et al. used multiplex PCR to simultaneously detect one to three exogenous genes, including cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium rhizogenes cochineal synthase gene terminator (NOS), Escherichia coli K12 strain (E. coli K12), Neomycin phosphotransferase U (NOS), and other exogenous genes, in the transgenic Petunia and the positive control plasmids. Neomycin phosphotransferase U (NptH) encoding gene. The results showed that multiplex PCR can not only improve the efficiency and reduce the cost of the assay, but also effectively prevent the occurrence of false-positive results.
(四) Gene rearrangements
The immunoglobulin (Ig) and T cell recetptor (TCR) loci contain a number of different V, D, and J gene fragments that are involved in the rearrangement process of early leukocyte differentiation.V-D-J fragment rearrangement is mediated by a recombinase complex in which the RAG1 and RAG2 proteins play an important role in the recombination signal sequences (RSS) by recognizing and cleaving DNA. The RAG1 and RAG2 proteins play an important role by recognizing and cleaving recombination signal sequences (RSS), which are located downstream of the V gene, at both ends of the D gene, and upstream of the J gene. Inappropriate RSS can reduce or even completely prevent rearrangements. van Dongen et al. successfully developed and standardized a multiplex PCR method to detect homologous cellular rearrangements of immunoglobulin and T-cell receptor genes, chromosomal aberrations t (11; 14) and t (14; 18). Of the 18 multiplex PCR reactions, 107 different primer pairs could be used for amplification. 14 Ig/TCR and 4 BCL1/BCL2 multiplexes proved to be well suited for cloning studies of lymphoproliferative defects with high sensitivity. In particular, the combination of IGH and IGK for suspected B-cell proliferation and TCRB and TCRG for suspected T-cell proliferation resulted in very high clonal detection rates.
(五) Resistance gene testing
The widespread use of antibiotics has led to an increase in the number of drug-resistant microorganisms, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci, and multidrug-resistant Mycobacterium tuberculosis. vancomycin resistant en-terococci and multidrug resistant Mycobacterium tuberculosis. Rapid detection of these pathogens and the corresponding antibiotic resistance is important for isolating patients and preventing further spread of the disease. Multiplex PCR can detect these antibiotic resistance genes simultaneously, saving time and providing high sensitivity and specificity.
Strommenger et al. applied multiplex PCR to simultaneously detect nine S. aureus resistance genes, including mecAy aacA ~ aphD, tetK, tetM, ermA, ermC, vatA, vatB, and vatC, and added an additional pair of primers to amplify S. aureus 16S rRNA as a positive control. The 16S rRNA of S. aureus was amplified as a positive control. The multiplex PCR results of 30 isolates of S. aureus were consistent with the resistance phenotypes obtained in the broth microdilution assay, which proved that multiplex PCR is a rapid, simple and accurate method for identifying antibiotic resistance and can be used in clinical diagnosis and epidemiological studies to monitor the transmission of resistance genes.
Multiplex PCR was also used to simultaneously detect the plasmid-mediated genes for chlordecone resistance, qnrA, qnrB and qnrS, and to screen 64 strains of ESBL-producing enterobacteria isolated in Kuwait.
(六) Microbial detection
Multiplex PCR can be used as a molecular detection and typing method for essentially all microorganisms, but its advantages can only be fully realized when used for the detection of difficult to culture or non-culturable microorganisms.
Multiplex PCR allows the selection of different primers to amplify different target fragments according to the needs. These combinations of amplification templates are generally chosen according to the following principles: different genes of the same microorganism (to minimize the occurrence of false-positive results); microorganisms of the same genus and different species, which are mainly used for molecular typing of microorganisms; and mixed microorganisms, which include microorganisms causing the same or similar symptoms, microorganisms having the same living environment, and microorganisms that are artificially placed together, such as combinations of war-wounded bacteria.
1. Detection of a microorganism (detection of virulence-related genes)
Staphylococcus aureus mainly produces staphylococcal enterotoxins (SE), toxic shock syndrome toxin (TSST), exfoliative toxins A and V. Lovseth et al. showed that nine enterotoxin genes, sae, seb, sec, sdl, see, seg, seh, sei, and sej, can be detected by multiplex PCR. Lovseth et al. used multiplex PCR to detect nine enterotoxin genes of Staphylococcus aureus, sae, seb, sec, sdl, see, seg, seh, sei, and sej, as well as toxic shock syndrome toxin (TSST), and the 16sRNA gene, and optimized the multiplex PCR to be able to correctly differentiate among the nine toxins.
Streptococcus suis is a pathogen that causes meningitis, arthritis, cardiomyocystitis, and pneumonia in piglets, mostly from 3 to 12 weeks of age, especially in newly weaned piglets. Streptococcus suis is widely distributed throughout the world and causes losses in pork production. Streptococcus suis infections have been difficult to control due to the lack of effective vaccines and sensitive diagnostic methods. To date, 35 serotypes have been identified based on podoconiotic antigens, of which 1/2, 1, 2, 7, 9 and 14 are the most common. However, tonsils can be infected by non-toxigenic Streptococcus suis and other streptococci, and it is difficult to differentiate them on the basis of colony morphology alone. To compensate for this, serotype-specific isolation in selective media and immunomagnetic bead techniques have been developed. However, to date, they have only been used for serotype 2 and serotype 1/2 typing. In addition, these methods are time-consuming, labor-intensive, and have low sensitivity. PCR methods allow for easy and specific differentiation of virulence-associated phenotypes and specific serotypes of Streptococcus suis, and the multiplex PCR methods based on these methods reduce the number of PCR reactions. Wisselink et al. developed two multiplex PCR systems that could be operated with 96-well plates and were able to detect six major serotypes and two virulence-related phenotypes of Streptococcus suis in pig tonsil specimens.
Guo X et al. used multiplex PCR to detect the toxin-related genes of enterohemorrhagic Escherichia coli (EHEC) O157:H7, including Shiga-like toxin genes (slt1 and slt2), from clinical isolates, A total of 85 O157:H7 strains were tested, and the detection rate of virulence genes was 56.5% (48/85), of which 79.2% (38/48) contained slt2, eaeA and hemolysin (hly), 16.6% (8/48) carried all four genes, and 4.2% (2/48) only had slt2 and hly genes. Unlike what has been reported in foreign literature, slt1 had a low carrier rate. The present study demonstrated that multiplex PCR can be a simple, rapid, specific and sensitive method for the detection of virulence genes.
2. Typing of microorganisms
(1) Homotyping of microorganisms: Since microorganisms are categorized into subspecies and strains below the species level, although there is a difference between the types of bacteria, it is not possible to differentiate them by phenotype alone, which poses a challenge to clinical diagnosis. The multiplex PCR method has been widely used as a method that can directly target the pathogen genes for typing.
Fujioka et al. developed two multiplex PCR reactions for the detection of nine virulence-related target genes of five types of diarrheagenic Escherichia coli (DEC). Reaction 1 consisted of five primer pairs: stxl, eaeA, invE, STp, and astA, and reaction 2 consisted of four primer pairs: stx2, aggR, STh, and LT. The two multiplex PCR reactions showed 100% specificity in identifying the relevant strains with no non-specific bands, and 51 DEC- and 38 astA-positive bacteria were detected from 683 suspected E. coli strains. . This study demonstrates that these methods can reduce the cost of multiplex PCR reaction reagents and contribute to the diagnosis of DEC in the clinical laboratory.
(2) Identification of microorganisms of the same genus and different species: Species-specific primers can be amplified to typify different species of microorganisms. Usually, genes such as virulence genes, degradation genes, enzyme genes, etc. are selected for a particular bacterium.
Alvarez et al. developed a multiplex PCR method for the detection and epidemiological typing of Salmonella, involving six primer pairs for the detection of the main serotypes and phage types of Salmonella in Spain, and one pair of primers as an internal positive control. When the method was applied to clinical fecal specimens, the sensitivity for the detection of the major serotypes Enteritidis and S. Typhimurium was 93%, the specificity 100% and the efficiency 98%, while the inhibition of the PCR reaction was low at 8%. cohen's kappa index showed that the multiplex PCR was in 95% agreement with the traditional culture-dependent method for Salmonella typing. Sharma et al. developed a multiplex PCR method for the detection of S. aureus toxin genes, combining universal and toxin-specific primers for the toxin genes, and a new DNA purification method, which allowed the detection of enterotoxin genes A to E from pure cultures within 3-4 h. The results of the multiplex PCR method used for the detection of Gluconobacter aureus isolates from multiple environments showed that the multiplex PCR method was comparable to the standard immunoassay method, and the results showed that the multiplex PCR method was comparable to the standard immunoassay method. The results showed that the multiplex PCR method was in 99% agreement with the standard immunotyping method, and it could not only amplify known toxin genes, but also could be used to detect toxin genes with new characteristics and unknown toxin genes.Panicker et al. applied multiplex PCR to detect pathogenic Vibrio spp. in littoral water and shellfish, and the genes selected included: vvh and viuB of Vibrio fragilis, ompU of Vibrio cholerae. toxR and hlyA, toxR and hlyA, ompU of Vibrio cholerae, vvh and viuB for Vibrio fragilis, ompU, toxR and hlyA for Vibrio cholerae, and tlh, tdh, trh and open reading frame 8 for Vibrio parahaemolyticus, which allowed the identification of all pathogenic strains and the typing of the three species.
(3) Identification of mixed microorganisms: Multiplex PCR can identify microorganisms that cause the same clinical symptoms or infect the same tissue or organ in a single reaction. Because different pathogenic microorganisms can cause the same or similar clinical symptoms, it is difficult to differentiate and identify them by clinical presentation alone. In some cases, pathogens that can be transmitted through the same route of infection, such as foodborne, waterborne, and seafoodborne pathogens, need to be tested simultaneously.
(1) Neurologic: Neurologic infections are a difficult diagnostic challenge for both clinicians and microbiologists. Herpesvirus infections can cause a variety of clinical symptoms, including encephalitis, myelitis, meningitis, etc. Bouquillon et al. have developed a multiplex PCR method that can simultaneously detect six human herpesviruses: herpes simplex virus (HSV) types 1 and 2, human cytomegalovirus (HCMV), varicella-zoster virus (varicella-zoster virus), and herpes zoster virus (varicella-zoster virus). HCMV, varicella-zoster virus (VZV), epstein-barr virus (EBV), and human herpes virus 6, suggesting that multiplex PCR is a rapid and reliable method for the diagnosis of herpes virus infection. The multiplex PCR method developed by Markoulatos et al. can simultaneously detect HSV-K, HSV-2, VZV, CMV, and EBV, etc. A total of 86 cerebrospinal fluid samples were tested. A total of 86 cerebrospinal fluid specimens were tested, and 9 positive specimens were detected, which accounted for 10.3% of the total specimens. Among them, 3 specimens were positive for HSV-1, which accounted for 3.5% of the total specimens; 4 specimens were positive for VZV, which accounted for 4.6% of the total specimens; 11 specimens were positive for HSC, which accounted for 1.16% of the total specimens; 1 specimen was positive for CMV, which accounted for 1.16% of the total specimens; and no specimens were positive for EBV. Multiplex PCR for the simultaneous detection of five different herpesviruses provides an early, rapid, reliable, and non-invasive diagnostic tool for guiding specific antiviral therapy, suggesting that multiplex PCR has significant clinical value. 156 immunocompetent patients and 44 immunodeficient patients). Of the 156 immunocompetent patients, 35% (55) were positive for enteroviruses and herpesviruses representing aseptic meningitis or encephalitis, and 41% (18) of the 44 immunodeficient patients, with herpesviruses being the predominant pathogen. Multiplex reverse transcription PCR is widely used and may be a particularly valuable rapid and sensitive diagnostic method for neurologic diseases.
② Causes diarrhea: The pathogens that cause diarrhea are mainly foodborne, so there are two main types of specimens for detecting diarrhea pathogens: food specimens and fecal specimens. Traditional testing methods include isolation and culture, biochemical identification, which is time-consuming and laborious, and it takes 4-7d to get the results.
The multiplex PCR method developed by Brasher et al. can detect E. coli, Salmonella typhimurium, Vibrio fragilis, Vibrio cholerae and Vibrio parahaemolyticus in shellfish, and the specific genes selected were uidA, cth, invA, ctx and tl genes. Among them, E. coli can indicate the degree of fecal contamination of the specimen, and the other four species are common pathogens in shellfish, and the optimized multiplex PCR method has a sensitivity of less than 101~102cfu, which is an effective, sensitive and rapid method for the detection of microbial pathogens in shellfish.Kim JS et al. selected the specific genes of Escherichia coli O157: H7, Salmonella, Staphylococcus aureus, Listeria monocytogenes and Vibrio parahaemolyticus. Kim JS et al. selected Shiga toxin (cytotoxin type II), femA (cytoplasmic protein), toxR (transmembrane DNA-binding protein), iap (invasion-associated protein), and invA (invasion protein A) of E. coli O157:H7, Salmonella, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus, respectively, and the results of the experiments demonstrated that these primers had good specificity, and the results of these primers could be obtained in 24 hours.
③ Causes respiratory symptoms: The traditional methods of diagnosing respiratory viral infections are cell culture and direct fluorescent antibody (DFA) assays. Multiplex reverse transcription PCR is a sensitive, specific and rapid method to detect viruses. The multiplex reverse transcription PCR (m-RT-PCR) method developed by Syrmis et al. used multiple virus-specific primers in combination with an enzyme-linked amplicon hybridization assay (ELAHA) for virus-specific gene sequences to detect 598 nasopharyngeal aspirates (nasopharyngeals) from patients with suspected respiratory tract infections. The enzyme-linked amplicon hybridization assay (ELAHA) was combined with the nasopharyngeal aspirate (NPA) assay to test 598 specimens from patients with suspected respiratory tract infections, with a method specificity of 100%. Compared with the m-RT-PCR-ELAHA method, the sensitivity of DFA was 79.7%, and that of culture amplified-DFA (CA-DFA) was 88.6%. Among the 598 NPA samples screened by m-RT-PCR-ELAHA, 3% were positive for adenovirus (ADV), 2% for influenza A, 0.3% for influenza B, 1% for parainfluenza type 1 (PIV1), and 1% for parainfluenza B. The sensitivity of DFA was 79% compared with that of m-RT-PCR-ELAHA. PIV1, 1% parainfluenza type 2 (PIV2), 5.5% parainfluenza type 3 (PIV3), and 21% respiratory syncytial virus (RSV). Compared with the DFA and CA-DFA methods, m-RT-PCR-ELAHA is sensitive, specific, and rapid, which is a significant improvement over the traditional methods for detecting respiratory viruses in clinical laboratories, and it is a method that can be used in daily clinical and laboratory operations.
Bellau-Pujol et al. established three multiplex reverse transcription PCRs for the simultaneous detection of 12 RNA respiratory viruses, including influenza viruses A, B, and C, human RSV, human metapneumoviruses (hMPV), PIV-1 2, 3, and 4, human coronaviruses OC43 and 229E, and rhinoviruses (hRV). rhinovirus, hRV). Compared to immunofluorescence and virus isolation methods, multiplex PCR is more sensitive, rapid, and capable of detecting a wider range of respiratory viruses.
Multiplex PCR can be combined with other methods to broaden its application. Combining multiplex PCR with real-time quantitative PCR breaks with the traditional scientific terminology, which usually describes the application of multiple oligonucleotide probes to distinguish between multiple amplicons. The limited variety of fluorophores available makes the application of this method difficult. RT-PCR is used to amplify RNA, and multiplex reverse transcription PCR in combination with multiplex PCR is also used to amplify RNA, mainly for the simultaneous detection of RNA viruses. Diaz de Arce et al. have established a new, highly sensitive, and specific multiplex reverse transcription PCR for the detection of classical swine fever and other swine fever viruses. Diaz de Arce et al. developed a new, sensitive and specific multiplex reverse transcription PCR for the simultaneous detection and typing of classical swine fever and other swine fever virus infections, with primers designed to amplify the non-coding region at the 5' end and the NS5B gene.
Materials and Instruments
Materials and Instruments
Equipment: PCR thermocycler, nucleic acid electrophoresis, gel imaging equipment, centrifuge, etc.
Reagents:
①Ultrapure water under high pressure (the purpose of high pressure is to inactivate DNase to avoid degradation of template DNA);
②PCR buffer (select the buffer corresponding to the polymerase used);
③4 dNTP mixtures (concentration of each dNTP was 2. 5 mmol/L).
④ Primers (primers were synthesized and diluted to 10 mmol/L with ultrapure water);
⑤ Heat-stable DNA polymerase (enzymes from different manufacturers and batches may vary);
⑥DNA template (try to use purified nucleic acids as templates).
Steps
The basic process of multiplex PCR can be divided into the following steps:
(一)Selection of target gene
Since multiplex PCR requires multiple pairs of primers in the same reaction system, and the templates have a direct impact on the analysis of the amplification results, the selection of amplification templates is of utmost importance. At the same time, the selection of the amplification region must be consistent with the purpose of the analysis, such as usually for disease-causing microorganisms, you need to select their conserved sequences, such as 16SRNA, or virulence genes, virulence-related genes, in order to prevent the detection of non-pathogenic mutants can not be interpreted; for the need to be typed for the object, you need to select the conserved sequences that are different from each other to be amplified; for the high degree of homology of sequences can be amplified using the same primers, but obtain the same primers to be amplified. For highly homologous sequences, the same primers can be used for amplification, but positive results need to be further determined by hybridization with specific probes or restriction enzyme cuts; for deletion analysis, amplification of exons is selected; for forensic identification of individual differences, amplification of highly polymorphic markers is selected; for transgenic testing, the transferred plant and animal loci are selected; and for gender identification, the loci specific to the X or Y sex chromosomes are generally selected.
For deletion analysis of genes containing multiple exons, the region with the wider hotspot of deletion or the region with dense deletion can be selected. Nearby exons can be amplified with primers that span both exons.
(二)Primer design
The purpose of primer design is to find a pair of suitable nucleotide sequences that can effectively amplify the template DNA sequence. Therefore, the quality of primers is directly related to the specificity and sensitivity of PCR. In order to determine the position of the primer, it is necessary to know the detailed DNA sequence information of the site where the selected gene primer binds to the template. In multiplex PCR, in order to ensure the amplification efficiency, all primer pairs must be optimized to similar amplification conditions. Therefore, the primer design of multiplex PCR should not only meet the general principles of PCR primer design, but also pay attention to the following issues:
(1)
the primers can not be complementary, especially to avoid the complementarity of the 3 』, in order to avoid the formation of dimerization, after the primer design for PCR amplification to test whether the primers are paired to form dimers;
(2)
the primers can not exist a large complementarity with the other amplification fragment and template, and there can not be a large complementarity between amplification fragment and templates. The primers should not be complementary to other amplified fragments and templates, and there should be no homology between the amplified fragments; (3) The length, (G+C) content and Tm value of the primers should be the same as far as possible; (4) There should be a certain degree of difference in the size of the fragment of the product of the amplification of the primers, in order to distinguish it by the electrophoresis method. Generally speaking, the larger the product fragments are, the greater the difference in length should be. This makes the design of multiplex PCR primers difficult.
(三)Nucleic acid extraction
Nucleic acid-dependent detection methods are affected by the purification of the target nucleic acid, and the degree of purification of nucleic acid determines the application of nucleic acid methods. The advantage of multiplex PCR is that it is rapid and systematic, and is mainly used for the detection of clinical specimens, including blood, tissues, feces, etc. At the same time, multiplex PCR requires a high level of nucleic acid templates, so the purification of nucleic acid extraction is particularly important, which is directly related to the amplification results.
Generally, templates prepared by boiling and lysing bacteria are sufficient for normal PCR reactions, but there are many problems with multiplex PCR reactions. If the conditions permit, multiplex PCR requires purified DNA as the template, which can ensure the smooth operation of multiplex PCR.
(四)Single-point PCR (also called single-primer PCR)
Before performing multiplex PCR, each primer pair must be subjected to single-point PCRO. Determine the conditions under which each primer pair is subjected to single-point PCR as listed in Table 14-1:
After the reaction is completed, compare the amplification results to ensure that all primers amplify the corresponding product bands under the same cycling conditions to ensure that the primers specifically amplify the corresponding target sequence.
(五) Multiplex PCR (mixing primers in equal concentrations)
Each pair of primers in the reaction system is mixed in equal concentration, and the concentration of other components in the system remains unchanged, apply the same reaction conditions as single-site PCR for simultaneous amplification of multiple sites, and adjust the multiplex PCR reaction system and the reaction conditions according to the amplification results.
Caveat
The reaction system and conditions of multiplex PCR are basically the same as those of single-point PCR, but it is not possible to accomplish it in one go, and it is necessary to ensure that each pair of primers in the multiplex PCR reaction can obtain sufficient amplification of the corresponding target, and the yield of the amplification products should be basically the same.
The factors affecting the amplification effect of multiplex PCR can be divided into two categories: reaction system and reaction conditions. The reaction system includes primer, buffer, Taq DNA polymerase, dNTP and MgCL, etc. The reaction conditions include annealing temperature, extension temperature, extension time, and the number of cycles.
1. Optimization of reaction systems
(1)Primer concentration: In the multiplex PCR system, there is a positive relationship between the amount of primer and the length of the amplified fragment, i.e., the longer the amplified fragment is, the more primers are needed, and the shorter the fragment is, the less primers are needed. At the same time, the effect of multiplex PCR amplification will be affected by the increase in primer interactions with each additional multiplex PCR amplification. In order to minimize this negative effect, the selection of appropriate inter-primer concentration is a key factor to ensure the success of multiplex PCR. The optimal concentration of each primer for a single PCR is determined by performing a single PCR amplification with different primers, and then following the multiplex PCR procedure, a multiplex PCR pre-test is performed with two or more primer pairs. Multiple pairs of primers increase the likelihood of primer complementarity at the 3' end, resulting in primer dimers; it is also possible that one amplicon may inhibit another, resulting in non-uniform amplification results. Even after optimizing the cycling conditions, the amplification products of some genes are still not obvious. Optimization of the multiplex PCR reaction system often results in little or no amplification of one or two target sites, while the other primers have good amplification efficiencies. To solve this problem, the relative concentration of primers can be adjusted appropriately to solve the problem by increasing the amount of primers for weak bands and decreasing the amount of primers for bright bands. Reducing the concentration of primer pairs with high amplification efficiencies is more likely to increase the yield of inefficient products than increasing the concentration of primers with low amplification efficiencies. Differences in primer concentrations in multiplex PCR reactions are generally obtained empirically. Adjustment of other reaction conditions, such as the concentration of Mg2+ or KCl in the reaction system, should be considered only if the primer concentration cannot be adjusted to meet the requirements.
In some cases, such as amplification products that are similar in sequence but different in length, the shortest product may amplify better, especially if some of the amplification fragments share a common primer. This can be avoided by starting several cycles of PCR with the primer for the long amplicon and then adding the primer for the short amplicon, or by lowering the primer for the short amplicon. Theoretically, the molar ratio of primer to target sequence should be at least 108:1. This amount of primer ensures that once the template DNA is denatured, it anneals to the primer and not to itself. Generally, the amount of primer should be at least 10 times the amount of template.
(2)Template and template concentration: The concentration and quality of DNA extracted from blood and fresh tissue can meet the requirements of multiplex PCR. For DNA extracted from bacteria, in order to minimize the influence of the sample on the amplification results, the amount of bacteria added to the lysate should not be too much, otherwise, due to incomplete lysis, the bacterial proteins will inhibit the activity of DNA polymerase, resulting in false-negative results.
To achieve stable results, the concentration of each sample should be determined, and in practice, the test is often performed on a sample basis. Therefore, the concentration of sample templates often varies, resulting in the amplification efficiency of each sample is not completely equal, and sometimes amplification failure occurs. In this case, the effective concentration of template should be considered and adjusted appropriately. The concentrations of template DNA from several different sources are as follows: mammalian genomic DNA 100 μg/mL; yeast genomic DNA 1 μg/mL; bacterial genomic DNA 0.1 μg/mL; plasmid DNA l-5ng/mL.
(3)Concentration of dNTP and MgCl2: dNTP and MgCl2 are important components in the PCR reaction system, so optimization of the concentration of dNTP and MgCl2 is also necessary.
In the PCR reaction, the concentrations of dNTP and MgCl2 are 200 μmol/L and 1.5 mmoI/L, respectively. The concentration of Mg2+ affects the specificity of the amplification to a great extent, and Mg2+ is generally proportional to the concentration of dNTP, and this ratio can be kept constant when adjusting the other conditions of the reaction after it has been determined. When the concentration of dNTP is kept constant, as the concentration of MgCl2 increases, the specificity of the reaction gradually increases, but when it increases to a certain extent, the reaction product is almost zero. dNTP and MgCl2 concentrations should be balanced in the PCR reaction, which may be due to the fact that dNTP is able to bind magnesium ions, and the TaqDNA polymerase needs free magnesium ions to exert its activity. In addition, dNTP master batch is very sensitive to repeated freezing and thawing, after 3~5 times of repeated freezing and thawing, the multiplex PCR reaction often can not be carried out well, and the amplification products are almost completely invisible, but this low stability of dNTP is not obvious in the amplification of single gene.
(4)Concentration of PCR buffer (KC1): If multiplex PCR systematically favors amplification of PCR products, the best option is to design a series of multiplex PCR experiments with a fixed Mg2+ concentration (1.5 mmol/L), increasing the concentration of KCI sequentially (1.0 to 2.0-fold), and then optimizing the primer for each primer pair. On the other hand, if it is preferred to preferentially amplify shorter products, the Mg2+ concentration should be gradually increased (up to 4.5 mol/L) while keeping the KC1 concentration unchanged. If the amplification efficiency of all products is low, try increasing the concentration of template and heat-stable DNA polymerase. If there is no improvement, increase the concentration of all primers exponentially and use a landing PCR with the replication and extension temperatures lowered by 2°C each.
Increasing the concentration of PCR buffer from IX to 2X significantly improves the efficiency of multiplex PCR, and this modification is more important than the modification of DMSO, glycerol, or BSA. Primers that produce long fragment amplification products generally amplify better at low salt concentrations, while primers that produce short fragment amplification products amplify better at high salt concentrations, which make it difficult to denature and unravel the long fragment amplification products.
(5)Taq DNA polymerase: As the number of primer pairs in the multiplex PCR system increases, the amount of dNTP and polymerase should be increased accordingly. The quality of the enzyme varies from manufacturer to manufacturer, and a concentration gradient experiment is needed to find the optimal amount of enzyme to use. The use of too much Taq DNA polymerase can lead to unbalanced amplification of different genes, a slight increase in background and a decrease in the specificity of the reaction. You can add 2U to a 25μl reaction volume and make slight adjustments based on the amplification results.
(6)Application of auxiliary agents (e.g. DMSO, glycerol, BSA): Adding 50~100 mL/L of DMSO or glycerol into the multiplex PCR reaction system can improve the efficiency and sensitivity of multiplex amplification, obtain more amplification products and reduce non-specific amplification. However, these auxiliaries may interfere with the experimental results on the other hand, because they have different effects on the amplification efficiency of each gene locus. DMSO at 50 mL/L may increase the amplification efficiency of one locus and decrease the amount of amplification products of another locus, while having no effect at all on some loci; similarly, DMSO may inhibit or promote non-specific amplification. Therefore, the effect of using these auxiliaries needs to be verified in each specific reaction system. The addition of BSA (e.g., 1.0 g/L) significantly improves multiplex PCR amplification efficiency. In some cases, BSA is more effective than DMSO and glycerol, but again, the effect of BSA needs to be verified experimentally.
Some researchers have suggested the use of DMSO and glycerol at concentrations ranging from 5% to 10% v/v to improve the efficiency and specificity of amplification, but the use of DMSO in multiplexed reactions can produce contradictory results. Therefore, the effect of the adjustment of DMSO and glycerol on the experimental results should be investigated according to the specific experiments.
2. Optimization of reaction conditioAns
Since there are multiple pairs of primers in a multiplex PCR reaction system and the amplified template fragments are of different lengths, the amplification efficiencies and speeds of each pair of primers are also different. Since the multiplex PCR reaction always follows the principle of preferential amplification of smaller fragments, the optimal PCR conditions required for each pair of primers are not the same (when designing a multiplex PCR with multiple pairs of primers, the PCR amplification conditions should be as consistent as possible for each pair of primers), so when selecting the conditions for the multiplex PCR amplification (especially the annealing temperature and time), we should try to choose the conditions favorable for the amplification of larger fragments.
(1)Annealing Time and Temperature: Among the cycle parameters, the main factors affecting the efficiency of multiplex PCR amplification are the recovery temperature and extension time. The strategy for setting the recovery temperature is similar to that for single PCR. The melting temperature (Tm) of the primers is calculated, and the complexation temperature Ta (Ta=Tm-5) is derived from this. The optimal Ta for multiplex PCR is then determined using the “stepwise introduction method”, i.e., the complexation temperature is adjusted according to the amplification results with each additional pair of primers until satisfactory results are obtained for each amplified gene fragment. Extension time is an important factor affecting amplification yield. As the number of amplified loci increases, the extension time should also be extended. It is more effective to increase the amplification yield by extending the extension time within the optimal Mg2+ and dNTP concentrations than by increasing the Mg2+ and dNTP concentrations alone.
The effect of annealing time on amplification efficiency is much less than the effect of annealing temperature, and lowering the annealing temperature by 4-6°C is necessary to amplify the same genes in a multiplex PCR. Specific amplification of other genes concurrently in the multiplex eliminates the effect of non-specific amplification, and similarly, genes that are amplified efficiently will result in lower amplification yields for genes that are amplified inefficiently.
(2)Extension temperature: High extension temperatures can reduce the amplification of certain genes, even with long annealing and extension times.
(3)Elongation time: In multiplex PCRs, where multiple genes are amplified simultaneously, the lack of enzyme and dNTP is a limiting factor, and more time is required to synthesize all the products. Increasing the extension time in multiplex PCR increases the amount of longer PCR products. It has also been shown that the amount of PCR products from all genes increases when the extension time is increased.
(4)Number of PCR cycles: The most significant increase in PCR product yield is around 25 cycles. Typically, 28 to 30 cycles are sufficient for a single reaction, and increasing the number of cycles to 60 has no significant effect on the amount of product.
In conclusion, the setting of multiplex PCR reaction conditions is a tricky issue, which is also the guarantee of the success of multiplex PCR. The general strategy is to carry out a single PCR reaction first, and set the conditions for each primer; then, add more primer pairs sequentially, and adjust the reaction conditions continuously until all primer pairs are able to amplify the target bands under the same conditions at the end.
Common problems
Common problems in multiplex PCR can be solved by changing the factors that affect the amplification of PCR.
1.If all product bands are weak
①Increase the extension time;
②Lower the extension temperature to 62~68;
③Gradually reduce the annealing temperature;
④Adjust the concentration of Tag DNA polymerase;
2. If the short fragment product is weakly banded
① Increase buffer concentration from IX to 1- 5X or 2X ;
② Reduce the annealing or extension temperature;
③Increase the amount of primers corresponding to weak bands;
3. if the long fragment product is weakly banded
① Increase the extension time;
② Increase the annealing and/or extension temperature;
③Increase the concentration of primers corresponding to weak bands;
④ Decrease the buffer concentration to 0.7X~0.8X while keeping the MgCl2 concentration 1.5-2 mmol/L unchanged;
4. In case of non-specific products
① If the non-specific product is a long fragment, increase the buffer concentration to 1.4X~2.0X;
② In case of short fragments, reduce the buffer concentration to 0.7X-0.9X; ② In case of short fragments, reduce the buffer concentration to 0.7X-0.9X.
③ Gradually increase the annealing temperature;
④ Reduce the amount of template and polymerase;
⑤Increase Mg2+ to 3 mmol/L, 6 mmcd/L, 9 mmol/L, and 12 mmol/L while keeping the dNTP concentration constant at 200 ptmol/L;
5. If none of the above has worked, the following can be tried
①Adding auxiliary BSA
②Add adjuvant DMSO or glycerol
③Re-compare primers to ensure that there are no interactions between primers;
④ Replace all solutions with new dNTP.
For more product details, please visit Aladdin Scientific website.
https://www.aladdin-e.com