Infection with these viruses may result in severe lower or upper respiratory tract disease requiring hospitalization. Multiplex PCR refers to the use of polymerase chain reaction to amplify several DNA sequences simultaneously. 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. These multiplex PCRs have a variety of formats but with a common aim of detecting and typing HPVs. These pathogens can be organ system specific or symptom specific with respect to the age of the patient and the epidemiological characteristics of these pathogens. In a multiplexing assay, more than one target sequence is amplified by using multiple primer pairs in the same reaction mixture. Whereas 12 of 21 (57.1%) heart transplant recipients showed CMV and/or HSV reactivation, HHV-6 was detected in 2 of 21 (9.5%) of the recipients and in 7 of 56 (12.5%) blood donors. In addition, the use of primers targeting consensus regions may allow recognition of new, undescribed human herpesviruses. from domestic dogs in the north of Iran M.T. Multiplex PCR for the diagnosis of Duchenne muscular dystrophy. These nonspecific products may be amplified more efficiently than the desired target, consuming reaction components and producing impaired rates of annealing and extension. The demands of transfusion medicine of high-speed, high-throughput screening will place great technical demands upon these procedures. Rapid simultaneous diagnosis of infections with respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction enzyme hybridization assay (Hexaplex). Because of the increased number of primers in the reaction, proper primer design is critical to the success of your multiplex reaction. The third consensus primer pair results in a PCR product of 550 bp with low-risk HPV infection (HPV-6 and -11). This should provide valuable information for monitoring and treating patients with HPV-related lesions, although the method lacks the capability to define individual HPV types, which limits its usefulness in epidemiological investigations. USA.gov. Application of the Multiplex PCR Method to Bacterial Isolates To test the feasibility of this method for clinical use, we isolated 60 colistin-resistant strains (43 isolates of E. coli and 17 isolates of Klebsiella pneumoniae ) from fecal samples of pigs and pets by selective medium containing 2 mg/L of colistin ( Supplementary Table S3 ). Detection of hog cholera and differentiation from other pestiviruses by polymerase chain reaction. DATE: 06/02/2017. Soc. Direct detection of Sabin poliovirus vaccine strains in stool specimens of first-dose vaccinees by a sensitive reverse transcription-PCR method. [ 7] spiked food samples (meat rinse … These properties include interregion differences in GC content, leading to preferential denaturation; higher binding efficiency because of GC-rich primers; differential accessibility of targets within genomes due to secondary structures; and the gene copy number within a genome. Multiplex PCR is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. Methods: Direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimens by a multiplex reverse transcription-PCR assay. Betaine can eliminate the base pair composition dependence of DNA melting. 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. READ THE PRODUCT PROFILE​ Seasonal variations in the rates of detection of the different organisms were noted. Multiplex PCRs for other transfusion-transmitted viruses have also appeared in the literature. Commercially available applications of multiplex PCR are as yet in their infancy. In patients for whom a diagnosis was confirmed at autopsy, the test was positive for HSV-1 or -2 for 19 patients (2%), producing a sensitivity and specificity of 100 and 99.6%, respectively. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. Multiplex PCR has also proved to be a valuable tool for genotyping HBV strains (84). The system uses three type-specific capture oligonucleotides linked covalently to a single microplate well and three type-specific multibiotinylated probes for detection. Given the advantages already demonstrated by the use of multiplex PCR along with the recent developments in this technology, future applications of PCR, when possible, should be aimed at constructing multiple detection systems in which a number of clinically and epidemiologically relevant pathogens (viruses, bacteria, parasites, and/or fungi) may be detected, characterized, and/or inevitably uncovered in a symptom- and/or system-specific manner. Prevalence and Molecular Differentiation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba hartmanni in Egypt. Two different genes of CMV were detected with a single-step multiplex PCR in clinical specimens from renal transplant recipients and other CMV-seropositive patients (11). The problem in evaluating all of the aforementioned multiplex PCR studies is that most have not included complete patient detail. Discrimination of Entamoeba moshkovskii in Patients with Gastrointestinal Disorders by Single Round PCR. https://en.wikipedia.org/wiki/Multiplex_polymerase_chain_reaction (99) developed a multiplex PCR utilizing primer pairs targeting a portion of thegag 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. The latter was based on the assumption that samples with small amounts of viral DNA are more likely to lead to the amplification of only one of the two targets. This technique requires two or more probes that can be distinguished from each other and detected simultaneously. Amplification biases that were strongly dependent on the choice of primers and dependent to a lesser extent on the templates have been described (100). This may be achieved through the utilization of primers with nearly identical optimum annealing temperatures (primer length of 18 to 30 bp or more and a GC content of 35 to 60% may prove satisfactory) and should not display significant homology either internally or to one another (17, 26, 43). (82) utilized three nested multiplex PCRs for detection of HSV and VZV; EBV and HHV-6; and members of the enterovirus group and echovirus type 22 and 23. This approach was evaluated on serial leukocyte DNA samples taken from 34 patients during the first 12 weeks post-renal transplantation, and it was concluded that findings of three consecutive tests in which both CMV targets were amplified were highly indicative of patients who had developed a very high load of CMV with a sensitivity of 100% and specificity of 88%. In one of these studies (73), the sensitivity of this multiplex PCR in 298 genital ulcer swab specimens to detect HSV,H. 1993;40(3):340–4. gondii DNA in 8 of 8 patients with toxoplasma encephalitis but in none without toxoplasmosis. 97th Gen. Meet. For example, Lam et al. These studies further highlight the difficulties of multiplex PCR and also provide substantial evidence for the importance of careful selection of oligonucleotide primers. In the group positive for VZV (28 patients), 16 patients had meningitis and 10 had encephalitis but clinical details were not available for 2 patients. For this reason, a number of 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. The test is carried out in two stages. The test produced 100% correlation with the results of the uniplex PCRs in 92 genital swabs (29 were positive for HSV, 16 were positive for HPVs, and one was positive for C. trachomatis). The assay provides rapid simultaneous detection, identification, and quantitation of these viruses in nasal wash specimens in a single test. We recommend this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys. A 166-bp PCR product with E. histolytica DNA, a 580-bp product with E. moshkovskii DNA and a 752-bp product with E. dispar DNA were generated in a single-round and multiplex PCR reaction. ( 24 ) developed five groups of multiplex nested-PCR assays that can detect 18 … 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 (32, 38, 40, 101). Simultaneous detection of both hepatitis B virus DNA and hepatitis C virus RNA using a combined one-step polymerase chain reaction technique. These tests proved suitable for routine use in a diagnostic laboratory and highlighted the importance of screening for more than one virus in patients with meningitis and encephalitis. Fewer publications discuss multiplex PCR (18, 28, 43). In the second round of amplification a series of three internal primer pairs are introduced, producing type-specific amplicons that were easily differentiated based on size upon gel electrophoresis. The test was sensitive and allowed monitoring of CMV infection by quantitation of CMV DNA. These specimens were also tested for H. ducreyi by culture which was reported to be negative for all specimens tested. The assay utilized three serotype-specific primer sets that map to the region of the poliovirus genome encoding the amino terminus of the VP1 capsid protein, a region known for its heterogeneity among the three Sabin poliovirus serotypes. HPVs were detected in samples from 69 (90.8%) of the 76 patients with cervical carcinoma, of which high-risk HPVs accounted for 82.9%. Clin Microbiol Rev. Multiplex PCR has the potential to produce considerable savings of time and effort within the laboratory without compromising test utility. Differentiation of polioviruses from nonpoliovirus enteroviruses in both clinical (stool) and environmental (sewage) specimens has been feasible using RT-multiplex PCR (29). The ability of the test to detect viral coinfection in both simulated specimens and clinical samples was also demonstrated. In a total of 195 stool samples collected from 26 vaccinees following administration of the first dose of the trivalent oral vaccine, the multiplex PCR was more sensitive than culture for the detection of poliovirus types 1, 2, and 3. ducreyi, and T. pallidum were 100, 98.4, and 91%, respectively, compared to 71.8, 74.2, and 81% by HSV culture,H. 1 2. Multiplex PCR involves the simultaneous amplification of two or more primer sets in a single reaction. A method for nested PCR with single closed reaction tubes. Single PCR assay with EntaF primer, combined with EhR, EdR, and EmR primers…, Multiplex PCR fragments amplified using EntaF primer combined with EhR, EdR, and EmR…, The single PCR assay of E. histolytica DNA with EntaF and EhR primers…, Multiplex PCR with EntaF primer combined with EhR, EdR, and EmR primers in…, NLM On the basis of amplicon size, the amplification assay differentiates the variable region of the hemagglutinin genes of the H1 and H3 subtypes of influenza viruses A and B and the counterpart (hemagglutinin, esterase, and fusion gene) of influenza virus C. Variants within the same type or subtype are then identified by heteroduplex mobility shift assay of the amplicons. Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs. Clipboard, Search History, and several other advanced features are temporarily unavailable. Alterations of other PCR components over those usually described for most uniplex PCRs have rarely improved the efficiency of the test. Detection of HCV and GBV-C/HGV infection by multiplex PCR in plasma samples of transfused subjects. The design of strain-specific polymerase chain reaction for discrimination of the raccoon rabies virus strain from indigenous rabies viruses of Ontario. The clinical diagnosis of genital ulcer disease in men. Hamzah Z, Petmitr S, Mungthin M, Leelayoova S, Chavalitshewinkoon-Petmitr P. J Clin Microbiol. Both the Hexaplex assay and virus culture were negative in the 40 nasal washes from the asymptomatic children. Viral DNA sequences were amplified in a single reaction, and the resulting amplicons were detected in real time by the hybridization of four differently colored, amplicon-specific detector probes called molecular beacons present within the same reaction tube. Although this adaptation is undoubtedly effective in most cases, it also considerably complicates the practical application of PCR. See this image and copyright information in PMC. Entamoeba moshkovskii and E. dispar are impossible to differentiate microscopically from the pathogenic species E. histolytica. The test was evaluated using clinical samples from 1,118 children with acute respiratory infections and was positive in 395 (35%) samples. Development of a multiplex PCR assay for the simultaneous detection and discrimination of HIV-1, HIV-2, HTLV-I and HTLV-II. -, Nazemalhosseini Mojarad E, Nochi Z, Sahebekhtiari N, Rostami Nejad M, Dabiri H, Zali MR, Kazemi B, Haghighi A. The primer and the probes utilized were evaluated using multiple virus isolates from each group and resulted in specific PCR products from all tissue culture-positive specimens without cross-reactivity among these seven viruses or with other common human respiratory viruses. Within the pre-S region nucleotide exchanges are observed that are partly correlated to the serological surface antigen subtypes. The authors concluded that the test is simple and reproducible and can be automated. 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 (119). The test was sensitive and specific for all 12 tissue culture-grown prototype viruses and when applied to respiratory specimens was more sensitive (41 of 112) than direct immunofluorescence or antigen detection following culture (34 of 112). Multiplex PCR: critical parameters and step-by-step protocol. These include PCRs which combined primers to produce a type-specific product size (97, 103) or those that utilized degenerate primers for HPV screening and strain-specific antisense primers for simple typing (56). Sunzeri et al. The PCR products were analyzed by hybridization in microtiter plates using virus-specific, biotinylated oligonucleotide probes. By utilizing equimolar concentrations of primers aligning the 3′ ends with one of two consensus regions within the herpesvirus DNA polymerase gene and the 5′ ends with the related or nonrelated sequences of each agent to be amplified, the first round of amplification yielded a 194-bp fragment indicating the presence of herpesvirus. Co-amplification of multiple regions of the HIV-1 genome by the polymerase chain reaction: potential use in multiple diagnosis. Multiplex PCR and its Applications 1. In a total of 2,233 CSF specimens from 2,162 patients, the PCR was positive in 147 specimens from 143 patients (6.6% of all patients) including enteroviruses (77 patients), HSV-1 (20 patients), VZV (7 patients), HSV type 2 (HSV-2) (6 patients), CMV (3 patients), JC virus (2 patients), and HHV-6 (1 patient). -, WHO/PAHO/UNESCO report. The technique has been used to screen for individual or symptom-associated viruses and examine associations of virus infection with disease. Multiplex polymerase chain reaction for human herpesvirus-6, human cytomegalovirus, and human β-globin DNA. This approach was considered to be a consensus multiplex PCR assay because all the primers are consensus and are multiplexed in the same PCR mixture for simultaneous amplification. Fotedar R, Stark D, Beebe N, Marriott D, Ellis J, Harkness J. Clin Microbiol Rev. Use of the polymerase chain reaction in the diagnosis of ocular disease. Rapid identification of nine microorganisms causing acute respiratory tract infection by single-tube multiplex reverse transcription-PCR: feasibility study. Amplicons were detected by liquid hybridization using 32P-end-labeled oligonucleotides. In addition, the methodology has proved to be a powerful tool for characterization of nonhuman viruses (50, 70, 85, 104, 114, 116). ducreyi culture, and dark-field microscopy for T. pallidum, respectively.  |  This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Ideally, all the primer pairs in a multiplex PCR should enable similar amplification efficiencies for their respective target. Basic requirements for designing optimal PCR primers. Mohammadzadeh A, Spotin A, Mahami-Oskouei M, Haghighi A, Zebardast N, Kohansal K. Parasitol Res. Development of a multiplex immunocapture RT-PCR assay for detection and differentiation of tomato and tobacco mosaic tobamoviruses. Of these, 37.5% were positive for RSV, 20% were positive for influenza A virus, 12.9% were positive for adenovirus, 10.6% were positive for enterovirus, 8.1% were positive for M. pneumoniae, 4.3% were positive for PIV type 3, 3.5% were positive for PIV type 1, 2.8% were positive for influenza B virus, and 0.2% were positive for Chlamydia trachomatis. The method amplified the corresponding virus in infected cells and in five clinical samples (HSV-1 PCR-positive CSF from a patient with encephalitis, HSV-2 PCR-positive CSF from a patient with meningitis, VZV culture-positive vesicular fluid from a patient with shingles, CMV culture-positive urine from a congenitally infected patient, and EBV PCR-positive peripheral blood from a patient with a lymphoproliferative syndrome). Indeed, a recent study (106) demonstrated the promise of multiplex PCR automation for both rapid and reliable screening of transfusion-transmitted viruses in donated blood and transplantable tissues. The percentages of specimens positive by the multiplex PCR for serotypes 1, 2, and 3 were 67.2, 82.6, and 53.8%, respectively, compared to 55.4, 64.1, and 27.7% by virus isolation. Thorough evaluation and validation of new multiplex PCR procedures is essential. Avian leukosis virus (ALV) subgroups A, B, and J are very common in poultry flocks and have caused serious economic losses in recent years. Multiplex polymerase chain reaction (Multiplex PCR) is a widespread molecular biology technique for amplification of multiple targets in a single PCR experiment. Of the 38 specimens, the multiplex PCR detected HSV in 31 specimens (81.6%) and HSV and T. pallidum in 1 specimen (2.3%) (coinfection). Investigation of vesicular rashes for HSV and HZV by PCR. 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 (A. C. Yeo, R. J. Cooper, D. J. Morris, and C. C. Storey, Abstr. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. The former often eliminates nonspecific reactions (particularly production of primer dimers) caused by primer annealing at low temperature (4 to 25°C) before commencement of thermocycling (21). These advances along with others to enhance sensitivity and specificity and to facilitate automation have resulted in the appearance of numerous publications regarding the application of multiplex PCR in the diagnosis of infectious agents, especially those which target viral nucleic acids. PCR drift is a bias assumed to be due to stochastic fluctuation in the interactions of PCR reagents particularly in the early cycles, which could arise in the presence of very low template concentrations (26, 68); variations in the thermal profiles of a thermocycler, resulting in unequal ramping temperatures; or simple experimental error. 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