Multiplex Polymerase Chain Reaction Primer Research Articles

NEW PRIMER DESIGN of MULTIPLEX POLYMERASE CHAIN REACTION (PCR) for DETECTION of Escherichia Coli and Salmonella Enterica in FOOD SAMPLE

Background: Multiplex PCR techniques are used to detect multiple pathogens at the same time simultaneously. In order to achieve the specificity and sensitivity of detection, it is important to optimize the multiplex PCR method properly. Developing suitable primers and optimizing PCR temperature to boost particular genes from pathogens are key factors for this optimization. Methods: For the simultaneous detection of Escherichia coli and Salmonella enterica in food samples, this research aims to develop a set of primers for multiplex polymerase chain reaction (PCR). Using Primer-BLAST software, the study utilizes specific primer designs for the phoA gene (Eschericia coli) and invA gene (Salmonella enterica). DNA isolation has confirmed successful extraction from the two bacterial samples. PCR was performed under different conditions, including Singleplex and Multiplex PCR, using two annealing temperatures of 53oC and 50oC. Results: The results showed that this method can effectively amplify target genes and indicate their specificity and reliability at both temperature levels. Given these results, it has successfully conducted multiplex PCR using the built primer pairs. Both annealing temperatures of 50oC and 53oC can be used to perform multiplex PCR to detect E. coli and Salmonella enterica in one PCR reaction. Conclusion: Through this research, we have created a new set of Multiplex PCR Primers and an optimized multiplex PCR technique for the simultaneous detection of E coli and Salmonella enterica in food samples. The rapid and simultaneous screening of E. coli and S. enterica, which contributes to improved food safety measures and pathogen detection in the food industry, is promising with this optimized PCR approach.

Multiplex primers employment for detection of rinderpest virus RNA by PCR

Given the high contagiousness and rapid spread of the rinderpest virus, timely and accurate diagnosis plays a key role in preventing epidemics and taking measures to control the disease. The study aims to evaluate the efficiency of using multiplex primers in the polymerase chain reaction method for the detection of rinderpest virus ribonucleic acid. The study included the analysis of samples such as blood serum and conjunctival swabs from 50 animals with clinical manifestations of the disease. The experiment involved the collection of clinical samples such as blood serum and conjunctival washings. The results demonstrate the high specificity of the developed primers. These primers stand out because they use two pairs of the same gene region with different variable sequences that are specific for all strains of the rinderpest virus. In the polymerase chain reaction, both pairs of primers are used simultaneously at equal concentrations and under the same conditions. An additional polymerase chain reaction performed using these primers at the optimal annealing temperature confirmed the successful amplification and specificity of the primers. The absence of dimers and nonspecific products in the negative control confirmed the purity and reliability of the results. Thus, these results demonstrate that the use of these multiplex polymerase chain reaction primers allows for the efficient detection of the ribonucleic acid of the rinderpest virus of different strains. The developed multiplex primers represent an innovative method for the diagnosis of rinderpest virus with the potential for use in veterinary practice and animal disease control

Identification of some microorganisms directly from clinical sample by multiplex PCR

Background and Aims: Infectious diseases are affecting people more often than before and that is the main cause of morbidity and mortality in developed and developing countries. Taking the necessary measures by diagnosing earlier and using appropriate antibiotics made significant contributions to the control of the infections. In our study we aimed to develop a method for the diagnosis of some of pathogen gram-negative bacteria by using multiplex PCR (Polymerase Chain Reaction) from a positive blood culture bottles. Methods: Extraction method was applied from the blood cultures in which growth was detected. After mono¬plex PCR experiments were performed and images were obtained, multiplex PCR experiments were performed. Considering the product sizes and primers for multiplex PCR, a quadruple bacterial set was prepared by matching Acinetobacter baumannii, Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa. Experiment conditions and tube concentration were adjusted by making serial trial experiments. Results: Multiplex-PCR-based methods have the advantage of defining multiple pathogens in a single PCR reaction. These are used for identifying respiratory infections, bacteraemia, and meningitis which are common bacterial and viral diseases. Type-specific genes and molecular techniques including identification of infectious diseases with nucleic acid amplification are promising tools for the verification of accurate diagnosis. The feasibility of this assay in routine diagnostic testing was evaluated using 146 positive blood cultures and 40 negative blood culture samples. Conclusions: Early diagnosis and intervention with effective antimicrobial therapy is important in reducing mortality that is associated with bloodstream infections.

Prevalence of herpes virus in chronic periodontitis patients with and without type 2 diabetes mellitus: A clinico-microbiological study.

Background:Unfavorable modifications of tooth and its surrounding structures result in periodontal complications. Viruses, in specific herpes virus, are known to increase disease severity in periodontal patients. Periodontitis is known to be more established in type 2 diabetes mellitus (DM2) patients. Hence, the detection of the viral load, its effect on the prevalence of periodontitis and the glycemic control status of patients are to be evidenced. The study aimed to reveal the association of herpes virus with periodontal parameters and its prevalence in DM2 patients.Materials and Methods:The cross-sectional study involved a total of 120 patients falling into three groups; Group I (healthy), Group II (periodontitis without DM2) and Group III (periodontitis with DM2) were subjected for sampling. Subgingival samples of periodontitis patients were tested for clinical parameters, and DNA extraction was performed. The presence of herpes virus (Epstein–Barr virus [EBV-1] and human Cytomegalovirus [HCMV]) was detected using multiplex polymerase chain reaction primers. Glycemic status of patients was recorded as glycosylated hemoglobin and scored accordingly. Chi-square test was performed to analyze the association between the categorical variables, and t-test/Mann–Whitney U-test/analysis of variance/Kruskal–Wallis test was used for continuous data.Results:Significant levels of EBV-1 were detected in Group III (n = 21, 52.5%), followed by Group II (n = 16, 40%) and Group I (n = 2, 5%) (P < 0.0001). HCMV was not detected. A significant association of EBV-1 to periodontal site-specific parameters was observed in Group II patients (P < 0.05). EBV-1 was predominant with poor glycemic status patients.Conclusion:This study revealed that the incidence of herpes virus infection in periodontal patients was higher in diabetic patients and the examined patients were prone to EBV-1 infections.

Improved multiplex PCR primers for rapid identification of coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) are opportunistic pathogens that are currently emerging as causative agents of human disease. Though CNS are widespread in the clinic and food, their precise identification at species level is important. Here, using 16S rRNA sequencing, 55 staphylococcal isolates were identified as S. capitis, S. caprae, S. epidermidis, S. haemolyticus, S. pasteuri, S. saprophyticus, S. warneri, and S. xylosus. Although 16S rRNA sequencing is universally accepted as a standard for bacterial identification, the method did not effectively discriminate closely related species, and additional DNA sequencing was required. The divergence of the sodA gene sequence is higher than that of 16S rRNA. To devise a rapid and accurate identification method, sodA-specific primers were designed to demonstrate that species-specific multiplex polymerase chain reaction (PCR) can be used for the identification of CNS species. The accuracy of this method was higher than that of phenotypic identification; the method is simple and less time-consuming than 16S rRNA sequencing. Of the 55 CNS isolates, 92.72% were resistant to at least one antibiotic, and 60% were resistant to three or more antibiotics. CNS isolates produced diverse virulence-associated enzymes, including hemolysin (produced by 69.09% of the isolates), protease (65.45%), lipase (54.54%), lecithinase (36.36%), and DNase (29.09%); all isolates could form a biofilm. Because of the increasing pathogenic significance of CNS, the efficient multiplex PCR detection method developed in this study may contribute to studies for human health.

Detection and Typing of Human Papilloma Viruses by Nested Multiplex Polymerase Chain Reaction Assay in Cervical Cancer.

Background:Cervical cancer is the leading cause of death from cancer in under-developed countries. Human papilloma virus (HPV) 16 and 18 are the most prevalent types associated with carcinogenesis in the cervix. Conventional Polymerase Chain Reaction (PCR), type-specific and consensus primer-based PCR followed by sequencing, Restriction Fragment Length Polymorphism (RFLP) or hybridization by specific probes are common methods for HPV detection and typing. In addition, some researchers have developed a multiplex PCR for simultaneous detection and typing of different HPVs.Objectives:The aim of the present study was to investigate the prevalence of HPV infection and its types in cervical Squamous Cell Carcinoma (SCC) using the Nested Multiplex PCR (NMPCR) assay.Patients and Methods:Sixty-six samples with histologically confirmed SCC were evaluated. Total DNA was isolated by phenol–chloroform extraction and ethanol precipitation. Nested multiplex PCR was performed with first-round PCR by GP-E6/E7 consensus primers for amplification of the genomic DNA of all known mucosal HPV genotypes and second-round PCR by type-specific multiplex PCR primer cocktails.Results:Human papilloma virus infection was detected in 78.8% of samples, with the highest prevalence of HPV 16 (60.6%) while concurrent infections with two types was detected in 10.6%.Conclusions:The NMPCR assay is more convenient and easy for analysis of results, which is important for fast diagnosis and patient management, in a type-specific manner.

High-throughput detection of genetically modified rice ingredients in foods using multiplex polymerase chain reaction coupled with high-performance liquid chromatography method

The aim of this study was to develop a method for simultaneous detection of a variety of genetically modified (GM) rice ingredients in foods using multiplex polymerase chain reaction (PCR) coupled with high-performance liquid chromatography (HPLC) assay. The following exogenous genes found in GM rice were selected as targets: CaMV35S, NOS, Cry1Ac, Bar, and Xa21. The endogenous gene PEPCex of rice was selected as an internal control. In brief, six pairs of primers for multiplex PCR were designed according to the specific region of CaMV35S, NOS, Cry1Ac, Bar, Xa21, and PEPCex, and following the optimization, a multiplex PCR assay was developed, and then the multiplex PCR products were subjected to HPLC analysis. The GM rice lines ShanYou 63, KeFeng 6, KangYou 97, and LLrice 62 were used as reference GM rice samples to evaluate the potential diagnostic capability of the method. Results demonstrated that the multiplex PCR-HPLC developed in this work was an efficient diagnostic method for simultaneous identification of the target genes with 0.15 ng/mL of high sensitivity, suggesting a better alternative for the rapid detection of many genetic modification events.

Novel multiplex polymerase chain reaction primer set for identification of Lactococcus species

The gram-positive bacterial genus Lactococcus has been taxonomically classified into seven species (Lactococcus lactis, Lactococcus garvieae, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolactis, Lactococcus chungangensis and Lactococcus fujiensis). This study aimed to develop a novel multiplex polymerase chain reaction (PCR) primer set for the identification of the seven lactococcal species, as well as to differentiate the two industrially important dairy subspecies, L. lactis subsp. lactis and L. lactis subsp. cremoris. A multiplex PCR primer set was designed based on the nucleotide sequences of the 16S rRNA gene of the seven lactococcal species. The specificity of the established one-step multiplex PCR scheme was verified using more than 200 bacterial strains, in which a complete sequence match was confirmed by partial sequencing of their 16S rRNA gene. The one-step multiplex PCR enables the identification and speciation of bacterial strains belonging to the genus Lactococcus and the differentiation of strains of L. lactis subsp. lactis and L. lactis subsp. cremoris. This work provides an efficient method for identification of lactococcal strains of industrial importance.

Nivalenol-Type Populations of Fusarium graminearum and F. asiaticum Are Prevalent on Wheat in Southern Louisiana

U.S. populations of the Fusarium graminearum clade cause head blight on wheat and barley and usually contaminate grain with the trichothecene mycotoxin deoxynivalenol (DON). Recently, however, individual nivalenol (NIV)-type isolates from the United States were described that belonged to either the newly described species F. gerlachii or the genetically distinct Gulf Coast population of F. graminearum sensu stricto (s.s.). Here, we describe the discovery of NIV-type F. graminearum s.s. populations that were found in high proportion (79%) among isolates from small-grain-growing regions of Louisiana. We genotyped 237 isolates from Louisiana with newly developed polymerase chain reaction (PCR) restriction fragment length polymorphism markers and multiplex PCR primers that distinguish among the three trichothecene types: the two DON types (15ADON and 3ADON) and NIV. These isolates were compared with 297 isolates from 11 other U.S. states, predominantly from the Midwest. Using Bayesian-model-based clustering, we discovered a southern Louisiana population of F. graminearum s.s. that was genetically distinct from the previously recognized pathogen population in the Midwest (MW15ADON population). Population membership was correlated with trichothecene type. Most isolates from the southern Louisiana population were of the NIV type, while the majority of the isolates from the Midwest were of the 15ADON type. A smaller proportion of isolates from Louisiana belonged to the previously described Gulf Coast population that was mostly of the 3ADON type. The NIV type was also identified in collections from Arkansas (12%), North Carolina (40%), and Missouri (2%), with the collections from Arkansas and North Carolina being small and unrepresentative. F. asiaticum was detected from the two southern Louisiana parishes Acadia and Alexandria. All identified 41 F. asiaticum isolates were of the NIV type. Greenhouse tests indicated that U.S. NIV types accumulated four times less trichothecene toxin than DON types on inoculated wheat. This is the first report of NIV-type populations of F. graminearum s. s. and F. asiaticum in the United States.

Design of multiplex PCR primers using heuristic algorithm for sequential deletion applications

The sequential deletion method is commonly applied to locate the functional domain of a protein. Unfortunately, manually designing primers for multiplex polymerase chain reaction (PCR) is a labor-intensive task. In order to speed up the experimental procedure and to improve the efficiency of producing PCR products, this paper proposes a multiplex PCR primers (MPCRPs) designer to design multiple forward primers with a single 3′-UTR reverse primer for extracting various N-terminal truncated mutants to quickly locate the functional domain of a cDNA sequence. Several factors, including melting temperature, primer length, GC content, internal self-complement, cross-dimerization, terminal limitation, and specificity, are used as the criteria for designing primers. This study obtains a near-optimal solution of primer sets that can be placed in as few test tubes as possible for one multiplex PCR experiment. Results Homo sapiens ribosomal protein L5, Homo sapiens xylosyltransferase I, and Bacteriophage T4 gene product 11 were used as test examples to verify efficacy of the proposed algorithm. In addition, the designed primers of Homo sapiens ribosomal protein L5 cDNA were applied in multiplex PCR experiments. A total of 48 forward primers and one reverse primer were designed and used to duplicate N-terminal truncated mutants of different lengths from the protein. The primers were classified into eight tube groups (i.e., test tubes) held within the same temperature range (53–57 °C), and the validity of the PCR products were verified using polyacrylamide gel electrophoresis (PAGE) with the functional domain correctly located. A software implementation of the proposed algorithm useful in assisting the researcher to design primers for multiplex PCR experiments was developed and available upon request.

A practical algorithm for multiplex PCR primer set selection

Selecting the minimum primer set with multiple constraints is an effective method for a successful and economical Multiplex Polymerase Chain Reaction (MP-PCR) experiment. However, there is no suitable algorithm for solving the problem. In this paper, a mathematical model is presented for the minimum primer set selection problem with multiple constraints. By introducing a novel genetic operator, we developed a parthenogenetic algorithm MG-PGA to solve the model. Experimental results show that MG-PGA can not only find a small primer set, but can also satisfy multiple biological constraints. Therefore, MG-PGA is a practical solution for MP-PCR primer design.

Augmented Therapy Based on PCR-MRD Levels of Children with Acute Lymphoblastic Leukemia: A Report from the Japanese Children's Cancer and Leukemia Study Group ALL 2000 MRD Pilot Study

Many studies have shown the presence of minimal residual disease (MRD) following therapy for childhood acute lymphoblastic leukemia (ALL) to be an important prognostic marker. We have also shown a significant relationship between survival outcomes in patients enrolled in the previous ALL 911 study and molecular MRD levels 5 weeks (time point 1, TP1) and 12 weeks (TP2) following the initiation of chemotherapy (Leukaemia and Lymphoma 2002; 43: 1001). The aim of this study was to evaluate if polymerase chain reaction (PCR)-based MRD assay is sufficiently dependable for tailoring therapy, and if augmented therapy can reduce MRD levels to those associated with a favourable outcome. The subjects were under 18 years of age, and had newly diagnosed precursor B or T-cell ALL. Patients below one year old and those with t(9;22) were excluded. Written informed consent was obtained from patients or their legal guardians. The ALL 941-based protocol (45thASH, San Diego, 2003) utilized PCR-based MRD assay using immunoglobulin & T-cell receptor gene rearrangements. MRD was detected by nested PCR, with screening of rearrangements using multiplex PCR primers as described previously (Leukaemia and Lymphoma 2002; 43: 1001). Patients were initially stratified into 3 risk groups (in ascending order: SR, HR, and HHR) according to leukocyte count and age at time of diagnosis. The MRD+/+ patients with levels ≥ 10−3 at both TP1 and TP2 received augmented therapy 14 weeks after initiation, and the remainder continued to receive the initial risk-adapted protocols. A total of 311 patients with a median age of 5.3 years (range 1.0–16.8) were eligible for this study. There were 4 (1.3%) non-responders and no deaths in induction. Of the 307 patients stratified, 169 (55%) were SR, 107 (35%) were HR, and 31 (10%) were HHR. The 2nd stratification by MRD level at TP2 was possible for 72.3% (222/307; insufficient DNA=28; missing time-points=25; no marker=32). Out of the 222 patients stratified, 125 (56.3%) were MRD−/−, 58 (26.1%) were MRD+/−, and 38 (17.4%) were MRD+/+. At the point of analysis, the median follow-up time was 63 months (range 33–89). The overall 5-year event–free survival (EFS) rate of the 307 patients was 80.1% (SE 2.5), higher than the EFS of the ALL941 study, which was 76.2% (SE 2.1) (p=0.167). The 5-year EFS rates according to the 1st stratification were 85.5% (SE 4) for SR, 76.1% (SE 4.5) for HR, and 64.6% (SE 9.2) for HHR, while the equivalent rates for the 2nd stratification were 87.0% (SE 3.1) for MRD−/−, 75.5% (SE 7.7) for MRD+/−, and 75.3% (SE 6.4) for MRD+/+. From the 95 patients whose MRD levels were measured at 5 consecutive points from TP1 to TP5 (5, 12, 18, 24, and 30 weeks after the start of therapy), 21 subjects with MRD+/+ received an augmented chemotherapy, and MRD levels became undetectable in 9 patients at TP3, 5 patients at TP4, and 4 patients at TP5. The corresponding cumulative 5-year relapse rates of those patients were 11%, 50%, and 50%, respectively. Thus, negative MRD status at TP3, but not at TP4 or TP5, seems to be associated with a favourable outcome. Our results confirm the strong performance of MRD-based treatment interaction in a multi-institutional study without adversely affecting the outcome in childhood ALL. Moreover, present findings suggest that an augmented therapy could reduce MRD to levels associated with a favourable outcome. To improve the applicability and accuracy of MRD assay, new MRD-PCR targets and RQ-PCR-based MRD detection are needed in subsequent studies.[Acknowledgment: This study was partly supported by grants from the Children's Cancer Association of Japan (CCAJ)].

Greene SCPrimer: a rapid comprehensive tool for designing degenerate primers from multiple sequence alignments.

Polymerase chain reaction (PCR) is widely applied in clinical and environmental microbiology. Primer design is key to the development of successful assays and is often performed manually by using multiple nucleic acid alignments. Few public software tools exist that allow comprehensive design of degenerate primers for large groups of related targets based on complex multiple sequence alignments. Here we present a method for designing such primers based on tree building followed by application of a set covering algorithm, and demonstrate its utility in compiling Multiplex PCR primer panels for detection and differentiation of viral pathogens.

Multiplex PCR design strategy used for the simultaneous amplification of 10 Y chromosome short tandem repeat (STR) loci.

The simultaneous amplification of multiple regions of a DNA template is routinely performed using the polymerase chain reaction (PCR) in a process termed multiplex PCR. A useful strategy involving the design, testing, and optimization of multiplex PCR primer mixtures will be presented. Other multiplex design protocols have focused on the testing and optimization of primers, or the use of chimeric primers. The design of primers, through the close examination of predicted DNA oligomer melting temperatures ( T(m)) and primer-dimer interactions, can reduce the amount of testing and optimization required to obtain a well-balanced set of amplicons. The testing and optimization of the multiplex PCR primer mixture constructed here revolves around varying the primer concentrations rather than testing multiple primer combinations. By solely adjusting primer concentrations, a well-balanced set of amplicons should result if the primers were designed properly. As a model system to illustrate this multiplex design protocol, a 10-loci multiplex (10plex) Y chromosome short tandem repeat (STR) assay is used.

A Greedy Algorithm for Minimizing the Number of Primers in Multiple PCR Experiments.

The selection of a suitable set of primers is very important for polymerase chain reaction (PCR) experiments. Most existing algorithms for primer selection are concerned with producing a primer pair for each DNA sequence. However, when all the DNA sequences of the target objects are already known, like the approximately 6,000 yeast ORFs, we may want to design a small set of primers to PCR amplify all the targets, which can then be resolved electrophoretically in a series of experiments. This would be quite useful, because decreasing the number of primers greatly reduces the cost of an experiment. This paper extends the problem of primer selection for a single experiment presented in Doi and Imai (Genome Informatics, 8:43-52, 1997) to primer selection for multiple PCR experiments, and proposes algorithms for the extended problem. The algorithms design primer sets one at a time. We extend the greedy algorithm for one PCR experiment in (Genome Informatics, 8:43-52, 1997) by handling amplified segments in DNA sequences that have been identified by primer pairs already selected and by changing the priorities in the greedy algorithm. This algorithm is applied to real yeast data. The number of primers equaled 85% of the number of identified DNA sequences, which represented more than 90% of all the target DNA sequences. This is 42% the number of primers needed for multiplex PCR. Furthermore, the length of each primer is less than half the length of multiplex PCR primers so the cost of producing the primers is reduced to 20% of the cost in the multiplex PCR case.

Detection of verotoxin-producing Escherichia coli O157:H7 by multiplex polymerase chain reaction.

We constructed primers for multiplex polymerase chain reaction (PCR) to detect verotoxin-producing Escherichia coli (VTEC) O157:H7. The multiplex PCR primers were designed from the sequence of the flagellin structural gene of Escherichia coli flagellar type H7 (GenBank under accession number L07388), and from the sequence of the rfbE gene of Escherichia coli O157:H7 (GenBank under accession number S83460). In addition to these primers, we used a primer pair reported by Karch and Meyer (J. Clin. Microbiol. 27: 2751-2757, 1989) to amplify various VT genes from VTEC. All of the examined specimens (18 isolates) of VT-producing E. coli O157:H7 showed a positive result by the multiplex PCR test with the three sets of primers. The sensitivity of detection for VT-producing E. coli O157:H7 was shown to be at least 3,000 cells per PCR tube.