Multiplex PCR-based Assay Research Articles

The Nonserotypeable Pneumococcus: Phenotypic Dynamics in the Era of Anticapsular Vaccines

Nonserotypeable pneumococci (NSP) are commonly carried by Australian Indigenous children in remote communities. The purpose of this study was to characterize carriage isolates of NSP from Indigenous children vaccinated with the seven-valent pneumococcal conjugate vaccine (PCV7) and to use these data to guide decisions on reporting of NSP. A total of 182 NSP were characterized by BOX typing, antibiogram analysis, and multilocus sequence typing (MLST) of common BOX types. NSP positive for the wzg capsule gene were analyzed by a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB-H) targeting capsule genes to determine the serotype. Among 182 NSP, 49 BOX types were identified. MLST of 10 representative isolates found 7 STs, including ST448 (which accounted for 11% of NSP). Non-penicillin susceptibility was evident in 51% of the isolates. Pneumococcal wzg sequences were detected in only 23 (13%) NSP, including 10 that contained an approximately 1.2-kb insert in the region. mPCR/RLB-H identified serotype 14 wzy sequences in all 10 NSP, and 1 also contained a serotype 3-specific wze sequence. Among the remaining 13 wzg-positive NSP, few belonged to the serotypes represented in PCV7. It appears that most NSP identified in Australian Indigenous children are from a true nonencapsulated lineage. Few NSP represented serotypes in PCV7 that suppress capsular expression. High rates of carriage and penicillin resistance and the occasional presence of capsule genes suggest a role for NSP in the maintenance and survival of capsulated pneumococci. To avoid the inflation of pneumococcal carriage and antibiotic resistance rates, in clinical trials, we recommend separate reporting of rates of capsular strains and NSP and the exclusion of data for NSP from primary analyses.

Establishment and Application of Multiplex-PCR for Wheat Glutenin Subunits Relative to Superior End-Use Quality

Wheat processing qualities are largely dependent on the number and composition of high-molecular-weight glutenin subunits (HMW-GS). It has been demonstrated that Ax1/Ax2*, Dx5, and overexpressed Bx7 (Bx7OE) are normally associated with superior end-use quality, especially dough strength. To date, Bx7OE allele has not been exploited widely in China wheat breeding program. With the optimization of reaction components and amplification condition, three pairs of PCR primers targeting for Ax1/Ax2*, Bx7OE, and Dx5 genes were used to establish a multiplex PCR for the purpose of enhanced molecular marker-assisted breeding with good reliability and low cost. The validation with wheat cultivars (lines) carrying known genes displayed that the developed multiplex PCR permitted the discrimination of these major HMW-GS in a single PCR reaction and agarose gel assay. A total of 89 wheat cultivars and advanced lines from Tibet, China were tested by the multiplex PCR-based assay. The results showed that the frequencies of Ax1/Ax2* and Dx5 were both 12.4%, and only 10.1% of the accessions possessed both Ax1/Ax2* and Dx5, whereas no accessions carried Bx7OE. Introduction of HMW-GS relative to good gluten quality should enhance the improvement of wheat quality in this region.

Identification of Salmonella spp. and Salmonella typhimurium by a multiplex PCR-based assay from poultry carcasses in Mashhad- Iran

Poultry meat has been identified as one of the principal foodborne sources of Salmonella. In this preliminary study the prevalence of Salmonella spp. and its typhimurium serovar contamination of broiler carcasses, were determined. Using the rinse test method, numbers of 60 samples, representing 20 broiler flocks, were collected from poultry carcasses after the chilling stage in the processing line at a commercial broiler slaughtering facility in Mashhad, Iran. The presence of Salmonella spp and Salmonella typhimurium in collected samples were assessed by performing the pre-enrichment and enrichment culture, followed by multiplex- PCR assay. The primers were selected from the invA and fliC genes, specific for the detection of Salmonella spp. and Salmonella typhimurium, respectively. In this study 8.3% and 1.6% of poultry carcasses were found to be contaminated with Salmonella spp and Salmonella typhimurium respectively. In order to provide a more accurate profile of the prevalence of Salmonella spp and Salmonella typhimurium in broiler carcasses, it is pertinent to use multiplex -PCR method that could be considered as an appropriate alternative to conventional culture method.

P40 A multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) for screening antimicrobial resistance genes

P40 A multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) for screening antimicrobial resistance genes

The development and evaluation of a single step multiplex PCR for simultaneous detection of Anopheles annularis group mosquitoes, human host preference and Plasmodium falciparum sporozoite presence

The Anopheles annularis group mosquitoes, subgenus Cellia Theobald (Diptera: Culicidae), includes five recognized species: An. annularis Van der Wulp, An. nivipes Theobald, An. pallidus Theobald, An. philippinensis Ludlow and An. schueffneri Stanton. From these five, the three most common species found in Orissa were considered for this study because of their remarkable vectorial and behavioral variation and the important role they play in malaria transmission. To identify and understand their role in malaria transmission we developed a single multiplex PCR-based assay. This assay included the detection of human blood feeding habit and Plasmodium falciparum sporozoite presence. Of the 186 An. annularis mosquitoes collected, morphological character-based identification showed that 94 were An. annularis, 54 were An. philippinensis and 38 were An. pallidus. However, the multiplex PCR assay confirmed that 91 were An. annularis, 56 were An. philippinensis and 39 were An. pallidus individuals after adjustments were made for misidentified specimens in the morphological method. Anopheles annularis and An. philippinensis were found positive for human blood, and two samples of An. annularis species were positive for P. falciparum sporozoites. This one-step PCR-based method constitutes a very powerful tool in large surveys of anopheline populations.

Simultaneous Identification of 14 Genital Microorganisms in Urine by Use of a Multiplex PCR-Based Reverse Line Blot Assay

The aim of this study was to develop and evaluate a sensitive method for the simultaneous identification of 14 urogenital potential pathogens. A multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to detect 14 urogenital pathogens or putative pathogens, namely Trichomonas vaginalis, Streptococcus pneumoniae, Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma parvum, U. urealyticum, Gardnerella vaginalis, Haemophilus influenzae, herpes simplex virus type 1 (HSV1) and HSV2, N. meningitidis, Mycoplasma hominis, M. genitalium, and adenovirus, using two species-specific primer pairs and probes for each. The method was validated using a reference strain or a well-characterized clinical isolate of each target organism and was found to be both sensitive and specific. The limits of detection for the mPCR/RLB assay varied among the 14 target organisms from 4.2 x 10(-1) to 7.0 x 10(-11) ng/microl of genomic DNA. There were no cross-reactions among any of the probes. This method was used to test 529 first-voided urine specimens from male patients with and without urethritis attending two Sydney sexual health clinics. One or more target species were detected in 193 (36%) subjects. Of 233 positive results, overall 216 (93%) were concordant between mPCR/RLB and a comparator method (culture and/or species-specific PCR), 9 were positive only by mPCR/RLB, and 8 were positive only by the comparator method. The mPCR/RLB method was an accurate, convenient, and inexpensive method for the detection of multiple potential pathogens in first-voided urine specimens from men.

Development of Tools for T Cell Repertoire Analysis (TCRB Spectratyping) for the Canine Model of Hematopoietic Cell Transplantation.

Analysis of the recombinatorial diversity of rearranged T cell receptor genes in mature T cells is an essential tool in the evaluation of immune status and immune reconstitution in hematopoietic cell transplantation studies. While the spectratyping technique is available in human clinical research and mouse models, the canine genome has not been sufficiently annotated to simply implement the assay on the dog TCRB locus. To this end, we have annotated by bioinformatics and experimentally all canine TCRBV segments, as well as TCRBD, TCRBC and most of TCRBJ segments. Of all 31 canine TCRBV found, 23 were functional and 8 were pseudogenes. A multiplex PCR-based assay was further designed to analyze the entire TCRBV spectratype in a set of 4 reactions each containing 4 to 5 V-segment specific forward primers and a common C-segment specific reverse primer. Direct sequencing of RT-PCR products confirmed that all amplified genes originated from predicted V-segments and that the designed V-specific PCR primers did not cross-react with other TCRBV family members. The usefulness of the spectratyping technique for canine model of transplantation was further demonstrated in analysis of T-cell repertoire reconstitution of irradiated dogs at different time points of recovery. Moreover, our simple and rapid V (J) annotation strategy relies on internet resources open to the general public and does not require specialized training in bioinformatics. It can be readily applied for de novo identification and mapping of TCRB gene families in other animal species where genome sequence drafts become available. [Display omitted]

A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples

Early detection of bloodstream infections (BSI) is crucial in the clinical setting. Blood culture remains the gold standard for diagnosing BSI. Molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients. Here, a multiplex real-time PCR-based assay for rapid detection of 25 clinically important pathogens directly from whole blood in <6 h is presented. Minimal analytical sensitivity was determined by hit rate analysis from 20 independent experiments. At a concentration of 3 CFU/ml a hit rate of 50% was obtained for E. aerogenes and 100% for S. marcescens, E. coli, P. mirabilis, P. aeruginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identification results with conventional microbiology for 1,548 clinical isolates yielded an overall specificity of 98.8%. The analytical specificity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identification in clinical sepsis.

Species identification in cell culture: a two-pronged molecular approach

Species identification of cell lines and detection of cross-contamination are crucial for scientific research accuracy and reproducibility. Whereas short tandem repeat profiling offers a solution for a limited number of species, primarily human and mouse, the standard method for species identification of cell lines is enzyme polymorphism. Isoezymology, however, has its own drawbacks; it is cumbersome and the data interpretation is often difficult. Furthermore, the detection sensitivity for cross-contamination is low; it requires large amounts of the contaminant present and cross-contamination within closely related species may go undetected. In this paper, we describe a two-pronged molecular approach that addresses these issues by targeting the mitochondrial genome. First, we developed a multiplex PCR-based assay to rapidly identify the most common cell culture species and quickly detect cross-contaminations among these species. Second, for speciation and identification of a wider variety of cell lines, we amplified and sequenced a 648-bp region, often described as the "barcode region" by using a universal primer mix targeted at conserved sequences of the cytochrome C oxidase I gene (COI). This method was challenged with a panel of 67 cell lines from 45 diverse species. Implementation of these assays will accurately determine the species of cell lines and will reduce the problems of misidentification and cross-contamination that plague research efforts.

Multiplex PCR-based real-time invader assay (mPCR-RETINA): a novel SNP-based method for detecting allelic asymmetries within copy number variation regions

We report the development of a real-time Invader assay combined with multiplex PCR (mPCR-RETINA), an SNP-based approach that can measure the allelic ratio in copy number variation (CNV) regions of a genome. RETINA monitors the real-time fluorescence intensity of each allele during the Invader assay and detects allelic asymmetries caused by genomic duplication/multiplication in heterozygous individuals. By combining mPCR-RETINA and real-time quantitative PCR that detects total copy number, we can estimate the copy number of each allele in CNV regions, which should be useful for investigating the functional significance of allele copy number with disease susceptibilities and drug responses. Also, mPCR-RETINA can efficiently refine the detailed structures of CNV regions. Due to the combination of RETINA with multiplex PCR, mPCR-RETINA requires a very small amount of genomic DNA for analysis (0.1-0.38 ng/locus). Additionally, mPCR-RETINA has clear advantages in its simple protocol and target-specific reaction, even in nonunique regions. We believe mPCR-RETINA will provide a significant contribution to identifying functional alleles in CNV regions.

Identification of Less-Common Streptococcus pneumoniae Serotypes by a Multiplex PCR-Based Reverse Line Blot Hybridization Assay

We developed a multiplex PCR-based reverse line blot (mPCR/RLB) assay to identify 50 uncommon pneumococcal serotypes. In combination with a previously described mPCR/RLB assay (3), all 90 pneumococcal serotypes can be identified individually (32 serotypes) or, because of predictable cross-reactions, to within small groups of two to five related serotypes (58 serotypes), which can be distinguished using serotype-specific antisera.

Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB)—a practical epidemiological and diagnostic tool

Combining multiplex PCR, sequentially, with reverse line blot hybridization (mPCR/RLB) is a convenient, objective way to identify up to 43 targets in 43 individual specimens simultaneously (using a 45-lane membrane format). It is more flexible and less expensive than DNA microarray. The number of targets is adequate for epidemiological and most clinical diagnostic applications; based on the same target (43) and specimen numbers (43), it is much more practical than conventional uniplex PCR (uPCR) and mPCR. We have used the protocol to identify and subtype bacteria, viruses and fungi and identify pathogens in clinical specimens; potentially, it could be used for many other applications, such as detection of mutations in, or identification of alleles of, eukaryotic genes. Development of each assay involves (i) careful primer and probe design, based on literature and sequence database searches, which are critical to success of the assay; and (ii) bench-top evaluation, using known samples, controls and dilution series, to confirm sensitivity, specificity and reproducibility. The assay takes about one and half working days to complete; about 4 h for the mPCR and 6 h for the RLB, including a total of 4 h 'hands-on' time.

Evaluation of a Multiplex PCR-Based Reverse Line Blot-Hybridization Assay for Identification of Serotype and Surface Protein Antigens of Streptococcus agalactiae

A 33-primer multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to identify Streptococcus agalactiae serotypes and surface protein antigen genes simultaneously. It was evaluated by using 551 clinical isolates. The mPCR/RLB assay was more sensitive than conventional serotyping, especially for protein antigen typing, but otherwise the results correlated well.

Multiplex PCR-Based Reverse Line Blot Hybridization Assay To Identify 23 Streptococcus pneumoniae Polysaccharide Vaccine Serotypes

We developed a multiplex PCR-based reverse line blot assay to identify 23 pneumococcal serotypes represented in the polysaccharide vaccine, using 334 well-characterized isolates, representing all 90 serotypes, and 268 "unknowns." The assay identified all target serotypes, but 11, which cross-react with 1 to 4 nonvaccine serotypes, could be distinguished using serotype-specific antisera.

Consecutive Use of Two Multiplex PCR-Based Assays for Simultaneous Identification and Determination of Capsular Status of Nine Common Neisseria meningitidis Serogroups Associated with Invasive Disease

We developed two Neisseria meningitidis multiplex PCR assays to be used consecutively that allow determination of the serogroup and capsular status of serogroup A, B, C, 29E, W135, X, and Y cnl-3/cnl-1-like-containing N. meningitidis isolates by direct analysis of the amplicon size. These assays offer a rapid and simple method of serogrouping N. meningitidis.

Reverse Line Blot Assay for Direct Identification of Seven Streptococcus agalactiae Major Surface Protein Antigen Genes

We developed a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) to detect the genes encoding members of the family of variable surface-localized proteins of Streptococcus agalactiae (group B streptococcus [GBS]), namely, Bca (Calpha), Rib, Epsilon (Epsilon/Alp1/Alp5), Alp2, Alp3, and Alp4, and the immunoglobulin A binding protein, Bac (Cbeta). We used the assay to identify these genes in a collection of well-characterized GBS isolates and reference strains. The results showed that mPCR/RLB avoids the common problems of cross-reaction and nontypability associated with protein typing using antisera. It is as sensitive as, but more practical than, separate gene-specific PCRs and would be suitable for large molecular epidemiological studies of GBS.

Development and Validation of a Multiplex PCR-Based Assay for the Upper Respiratory Tract Bacterial Pathogens Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis.

Background: Conventional simplex polymerase chain reaction (PCR)-based assays are limited in that they only provide for the detection of a single infectious agent. Many clinical diseases, however, present in a nonspecific, or syndromic, fashion, thereby necessitating the simultaneous assessment of multiple pathogens. Panel-based molecular diagnostic testing can be accomplished by the development of multiplex PCR-based assays, which can detect, individually or severally, different pathogens that are associated with syndromic illness. As part of a larger program of panel development, an assay that can simultaneously detect Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis was developed. These organisms were chosen as they are the most common bacterial pathogens associated with both the acute and chronic forms of otitis media; they are also responsible for a high percentage of sinus infections in both children and adults. In addition, H. influenzae and S. pneumoniae are commonly associated with septic meningitits. Methods and Results: Multiple individual PCR-based assays were developed for each of the three target organisms which were then evaluated for sensitivity and specificity. Utilizing the simplex assays that met our designated performance criteria, a matrix style approach was used to develop a duplex H. influenzae-S. pneumoniae assay. The duplex assay was then used as a single component in the development of a triplex assay, wherein the various M. catarrhalis primer-probe sets were tested for compatibility with the existing assay. A single-step PCR protocol, with species-specific primers for each of the three target organisms and a liquid hybridization-gel retardation amplimer detection system, was developed, which amplifies and then discriminates among each of the amplification products according to size. This assay is able to detect all three organisms in a specific manner, either individually or severally. Dilutional experiments indicate a detection limit of 10 femtograms (fg)(6-7 genomic equivalents) or less of genomic DNA for each of the three microorganisms regardless of the presence of irrelevant DNA. Conclusions: The reliance on individual, robust, species-specific primers and the avoidance of a nested PCR approach make this bacterial multiplex assay suitable for use in the clinical laboratory. This assay has proved useful in both research and patient care applications.

Characterization of simple sequence repeat variants linked to candidate genes for behavioral phenotypes

Simple sequence repeats (SSRs) have traditionally been used as markers in gene mapping studies and typing for forensic purposes. Recently there has been some speculation that this type of genetic variation also plays a more direct role in influencing gene expression and hence complex phenotypic outcomes such as human behavior. For this reason it is interesting to investigate SSRs linked to candidate genes for various complex phenotypes. An economical multiplex PCR-based assay was designed to simultaneously genotype individuals at 15 loci across 10 candidate genes for human behavioural phenotypes, including seven loci previously unreported in Caucasians (five unreported in any population). All loci were tested for Hardy-Weinberg equilibrium and for two-locus Linkage Disequilibrium. Ewens-Watterson neutrality testing indicated possible selection at a previously unreported DRD2 locus.

A multiplex PCR for simultaneous detection and differentiation of North American serotypes of bluetongue and epizootic hemorrhagic disease viruses

In the present study, a multiplex RT-PCR-based assay for simultaneous detection and differentiation of North American serotypes of bluetongue (BT) virus (BTV) and epizootic hemorrhagic disease (EHD) virus (EHDV) in cell culture and clinical samples was developed. Two pairs of primers (B1 and B4) and (E1 and E4) were designed to hybridize to non-structural protein 1 (NS1) genomes of (BTV-11) and (EHDV-1), respectively. The multiplex PCR-based assay utilized a single tube-PCR amplification in which EHDV and BTV primers were used simultaneously in a multiplex format. The BTV primers generated a 790 base pair (bp) specific PCR product from RNA samples of North American BTV serotypes 2, 10, 11, 13 and 17; whereas EHDV serotypes 1 and 2 or total nucleic acid extract from non-infected baby hamster kidney (BHK) cells failed to demonstrate the 790 bp specific BTV PCR product. Likewise, the EHDV primers produced a 387 bp specific PCR product from RNA samples of EHDV serotypes 1 and 2, but not from BTV serotypes 2, 10, 11, 13, 17 or from total nucleic acid extract of BHK cell controls. Two pairs of nested primers (B2 and B3) and (E2 and E3), internal to the annealing sites of primers (B1and B4) and primers (E1 and E4), produced a 520 bp specific BTV and a 224 bp specific EHDV PCR product from BTV and EHDV first amplification products, respectively. These nested amplifications increased the sensitivity of the PCR assay and confirmed the specificity of the first amplified EHDV or BTV PCR products. The described multiplex RT-PCR-based assay could be used to facilitate rapid detection and differentiation of North American BTV and EHDV serotypes and to provide a valuable tool to study the epidemiology of these orbivirus infections in susceptible animal populations.

Use of a Multiplex PCR-based Assay to Differentiate Campylobacter jejuni and Campylobacter coli Strains Isolated from Human and Animal Sources

Use of a Multiplex PCR-based Assay to Differentiate Campylobacter jejuni and Campylobacter coli Strains Isolated from Human and Animal Sources