Single Polymerase Chain Reaction Reaction Research Articles

A Multiplex PCR System of Novel Microsatellite Loci for Population Genetic Application in Walnuts.

Multiplex polymerase chain reaction (PCR) of microsatellite loci allows for simultaneous amplification of two or more pairs of primers in a single PCR reaction; hence, it is cost and time effective. However, very few attempts have been reported in non-model species. In this study, by combining a genome-based de novo development and cross-species application approach, a multiplex PCR system comprising 5 PCR reactions of 33 microsatellites consisting of 26 novel genomic and 7 literature-sourced loci was tested for polymorphisms, cross-species transferability, and the ability to assess genetic diversity and population structure of three walnut species (Juglans spp.). We found that the genome-based approach is more efficient than other methods. An allelic ladder was developed for each locus to enhance consistent genotyping among laboratories. The population genetic analysis results showed that all 33 loci were successfully transferred across the three species, showing high polymorphism and a strong genetic structure. Hence, the multiplex PCR system is highly applicable in walnut species. Furthermore, we propose an efficient pipeline to characterize and genotype polymorphic microsatellite loci. The novel toolbox developed here will aid future ecology and evolution studies in walnut and could serve as a model for other plant species.

Multiplex PCR for Detection of Exfoliative Toxins, and Toxic Shock Syndrome Toxin 1 Genes in MRSA Strains of Clinical Staphylococcus aureus Isolates

This research approaches to investigate toxin gene content of the methicillin resistant Staphylococcus aureus strains that are clinically collected from human skin infection cases by performing multiplex polymerase chain reaction (PCR) technique (irrespective of whether the strain produces the toxin or not) and for that a single PCR reaction was employed, two pairs of exfoliative toxin and one pair toxic shock syndrome toxin-1. Amplicon sizes ranged between 93, 226 and 326bp, to facilitate electrophoretic separation. Results of the current study had revealed that the major gene detected was tst found in six isolates. Multiple toxin gene combinations were also observed. This work was conducted at several institutions including Tikrit Teaching Hospital’s lab; college of science biology department labs/ Tikrit University; and the Biotechnology Research Centre/ Al-Nahrain University.

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.

An accurate genetic assay to identify human neutrophil antigen 2 deficiency.

We aimed to develop accurate and user-friendly genetic assays to identify the inherited neutrophil antigen-2 (HNA-2) deficiency in humans. HNA-2 is one of the most important neutrophil antigens implicated in a number of human disorders. HNA-2 deficiency or HNA-2 null is a common phenotype observed in 3%-5% Americans. HNA-2 null individuals are at risk to produce isoantibodies (or alloantibodies) that play important roles in transfusion-related acute lung injury, immune neutropenia, and bone marrow graft failure. We previously demonstrated that the CD177 coding SNP 787A > T (c.787A > T) is the most important genetic determinant for HNA-2 deficiency. However, reliable genetic assays are not available for routine clinical laboratory application up to now. A novel polymerase chain reaction (PCR) strategy was used to determine genotypes of the CD177 SNP c.787A > T. In the simplified PCR assay, all allele specific primers and internal control primers were included in the same reaction, which ensures reliability of the assay. In addition, a novel high-throughput nested TaqMan assay was developed to determine genotypes of c.787A > T for large population genetic analysis of HNA-2 deficiency. CD177 SNP c787A > T genotypes of 396 subjects were 100% concordant among the single PCR reaction method, the nested TaqMan assay, and Sanger Sequencing analysis. Out of 396 subjects, all 18 donors with the CD177 STP homozygous genotype were HNA-2 null. The novel PCR-based genotyping assay is accurate to identify HNA-2 deficient individuals and is suitable for clinical laboratories. In addition, the innovative high-throughput nested TaqMan assay will be useful for large-scale population screens and genetic studies of HNA-2 deficiency.

A new Trypanosoma cruzi genotyping method enables high resolution evolutionary analyses.

BACKGROUNDTrypanosoma cruzi is an important human pathogen in Latin America with nearly seven million people infected. It has a large degree of genetic diversity, classified into six discrete typing units (DTUs), which probably influences its physiological behavior and clinical manifestations. Several genotyping methods are available, with distinct performance on easiness, cost, resolution and applicability; no method excels in all parameters.OBJECTIVES AND METHODS To devise a molecular method for T. cruzi genotyping, based on polymerase chain reaction (PCR) amplification of a single target with multiple copies in the nuclear genome by large scale sequencing. We have applied this method to 29 T. cruzi isolates, comprising all described DTUs.FINDINGS We were able to classify all samples into sub DTU level with high robustness. Evolutionary relationship between DTUs were ascertained, suggesting that TcIII and TcIV DTUs are non-hybrid, and DTU IV is more similar to the common ancestral.CONCLUSION As the TS-LSS method is based on a single PCR reaction, comprising several copies of the target, it is probably useful for clinical samples, when the amount of DNA is a limiting factor. As large scale sequencing systems become more common, the TS-LSS method can be increasingly applied for T. cruzi genotyping.

An Improved Multiplex Polymerase Chain Reaction (PCR) Assay for the Identification of Mosquito (Diptera: Culicidae) Blood Meals.

The analysis of vertebrate blood meals serves as an integral component of vector incrimination studies where feeding preferences and host associations influence vector-borne disease transmission. Diagnostic polymerase chain reaction (PCR)-based techniques have been widely used to determine host associations, yet applications for Culex (Diptera: Culicidae), which feed primarily on bird populations, have been limited by multistep PCR techniques that approach each potential host species singly. As a result, we have developed a multiplexed primer set targeting mitochondrial cytochrome b sequences that can distinguish human, bird, and mammalian host blood meals in a single PCR reaction, an improvement over previous analyses relying on single primers or other multiplex primer approaches through the inclusion of avian primers. To validate this new methodology, we demonstrate its application on blood samples as well as field-collected Culex samples. Although designed for applications with mosquito vectors, this multiplex PCR assay is not mosquito-specific, and should serve as a valuable tool for identifying the blood meals of other blood-feeding arthropods, contributing greatly to the study of vector-borne disease.

A novel 13-plex STR typing system for individual identification and parentage testing of donkeys (Equus asinus).

Previous studies investigating donkey parentage and genetic diversity used horse-specific multiplex systems. However, several mis-allele and null-allele issues were found with some of the horse primers when used in donkeys. In 2017, the International Society for Animal Genetics (ISAG) recommended 13 dinucleotide short tandem repeats (STRs) (AHT4, ASB23, HMS2, HMS3, HMS6, HMS7, HMS18, HTG7, HTG10, TKY297, TKY312, TKY337 and TKY343) as a core panel that should be used to identify individuals and to test for parentage in donkeys. To date, no single multiplex STR typing system containing all 13 donkey STRs recommended by the ISAG has been reported. To establish a novel and donkey-specific multiplex STR typing system containing all 13 recommended STRs. Assay development and validation in field population. Primers for seven of the STRs were redesigned and conditions for polymerase chain reaction (PCR) were optimised. We analysed the allele sequences, sensitivity, species-specificity and stutter ratios of this new system. A 13-plex STR typing system for donkey was established. A full profile could be generated from a single PCR reaction using as little as 5ng of DNA template with the 13 pairs of primers labelled with fluorescent dyes. An allele ladder, containing 101 alleles from the 13 STRs, was generated. No full genotype profile was generated with these primers if DNA from humans, or 11 other commonly encountered animals, was used. Genotypes could be generated for the horse and horse-donkey hybrids (mule and hinny). Stutter ratios and population genetic parameters were calculated based on samples from 150 donkeys. The combined probabilities of paternity exclusion for this system were 0.988907326 (CPEduo) and 0.999665018 (CPEtrio). This system cannot detect sex. Our results indicate that our donkey-specific 13-plex STR typing system is sensitive, species-specific and robust for individual identification, paternity testing and population genetic analysis in donkeys, and has potential forensic applications.

Authentication of the Herbal Medicine Angelicae Dahuricae Radix Using an ITS Sequence-Based Multiplex SCAR Assay.

The accurate identification of plant species is of great concern for the quality control of herbal medicines. The Korean Pharmacopoeia and the Pharmacopoeia of the People’s Republic of China define Angelicae Dahuricae Radix (Baek-Ji in Korean and Bai-zhi in Chinese) as the dried roots of Angelica dahurica or A. dahurica var. formosana belonging to the family Apiaceae. Discrimination among Angelica species on the basis of morphological characteristics is difficult due to their extremely polymorphic traits and controversial taxonomic history. Furthermore, dried roots processed for medicinal applications are indistinguishable using conventional methods. DNA barcoding is a useful and reliable method for the identification of species. In this study, we sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes in A. dahurica, A. dahurica var. formosana, and the related species A. anomala and A. japonica. Using these sequences, we designed species-specific primers, and developed and optimized a multiplex sequence-characterized amplified region (SCAR) assay that can simply and rapidly identify respective species, and verify the contamination of adulterant depending on the polymerase chain reaction (PCR) amplification without sequencing analysis in a single PCR reaction. This assay successfully identified commercial samples of Angelicae Dahuricae Radix collected from Korean and Chinese herbal markets, and distinguished them from adulterants. This multiplex SCAR assay shows a great potential in reducing the time and cost involved in the identification of genuine Angelicae Dahuricae Radix and adulterant contamination.

Optimizations of high throughput multiplex polymerase chain reaction with simple sequence repeat markers for genotyping of common walnut populations (<i>Juglans regia</i> L.)

Multiplex polymerase chain reaction (PCR) allows amplification of two or more pair of primers in parallel for amplification of multiple target sequences in a single reaction tube. In this study, we combined existing simple sequence repeat (SSR) markers (nuclear microsatellites) in the novel combination of multiplex PCR to study the population genetics of common walnut from Croatia. From twenty one tested SSR markers, eleven produced satisfactory results in one multiplex PCR. Population genetic results achieved from 15 samples of Croatian common walnut showed moderate genetic variability (average value: He 0.473; Ho 0.568). Our multiplex PCR allowed cost effective work concerning chemicals, plastic ware, device, and working time producing optimal results. The optimized multiplex PCR represented the best combination of eleven SSR primers for genotyping common walnut in a single PCR reaction.

Simultaneous detection of pyrethroid, organophosphate, and cyclodiene target site resistance in Haematobia irritans (Diptera: Muscidae) by multiplex polymerase chain reaction.

The horn fly, Haematobia irritans irritans (L., 1758) (Diptera: Muscidae), is an important pest that causes significant economic losses to the livestock industry, but insecticide resistance in horn fly populations has made horn fly control increasingly difficult to achieve. In this study, we developed a multiplex polymerase chain reaction (PCR) assay to simultaneously detect target site resistance to pyrethroids (kdr mutation), organophosphates (G262A acetylcholinesterase mutation), and cyclodienes (Rdl mutation) and used the new procedure to follow the progression of these three mutations after exposure to different insecticide pressure. We assayed flies collected at the Macon Ridge research station, Winnsboro, LA, from 2008 to 2012. The multiplex PCR showed robust results in all our assays. The kdr mutation remained at high frequencies during all years, even after 4 yr with no use of pyrethroids. The G262A acetylcholinesterase mutation fluctuated from 7.5 to 23.8% during the studied years, while the Rdl mutation was rare in 2008, 2009, and June 2010, and then significantly increased after the first use of endosulfan. The possibility of screening for all the known target site resistance mutations in a single PCR reaction makes the multiplex PCR a useful and affordable tool that can be used to help diagnose insecticide resistance.

Molecular Identification ofAedes triseriatusandAedes hendersoniby a Novel Duplex Polymerase Chain Reaction Assay

Aedes triseriatus is the principal vector of La Crosse virus (LACv), which is the most common cause of pediatric arboviral encephalitis in North America. Here we report a novel species-specific polymerase chain reaction (PCR) assay that differentially identifies Ae. triseriatus and Ae. hendersoni. Because these 2 sibling species differ in their abilities to transmit LACv, accurate identification is critical for surveillance, research, and control programs. This duplex assay can detect the presence of both species in a single PCR reaction and is therefore simpler and faster than previously reported methods.

Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples

Cyclospora, Cystoisospora, and Microsporidia are eukaryotic enteropathogens that are difficult to detect in stool samples because they require special stains and microscopy. We developed a multiplex polymerase chain reaction (PCR) reaction with 4 primer sets to amplify Cyclospora cayetanensis, Cystoisospora belli, Enterocytozoon bieneusi, and Encephalitozoon intestinalis. Detection of the amplicon is through specific probes coupled to Luminex beads. Sensitivity of the assay was evaluated using Encephalitozoon intestinalis spores and revealed detection of 10 1 spores spiked into stool. No cross-reactivity was observed. We evaluated the assay on diarrheal specimens from Thailand, Tanzania, Indonesia, and the Netherlands that had been previously tested by microscopy, and the assay yielded 87–100% sensitivity and 88–100% specificity. Microscopy-negative/PCR-positive samples had lower Luminex values, suggesting they were true but with lower burden infections. In summary, this is a convenient single PCR reaction that can detect Cyclospora, Cystoisospora, and Microsporidia without the need for cumbersome microscopic analysis.

Molecular Detection of Autosomal-Dominant Feline Polycystic Kidney Disease by Multiplex Amplification Refractory Mutation System Polymerase Chain Reaction

Feline autosomal-dominant polycystic kidney disease (ADPKD), with its characteristic growth of fluid-filled cysts of different sizes, is the most prevalent inherited genetic disease of cats. The point mutation (C-->A transversion) in exon 29 of the PKD1 gene is known to contribute to ADPKD development and can thus serve as a target for the molecular genetic diagnosis of ADPKD. To this end, a simple amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) was designed with 3 primers: 2 forward primers specifically targeting either the mutant or normal allele, and 1 universal reverse primer for amplification of both alleles. The new method was tested on the DNA from 35 feline blood samples, which included 15 mutant cats and 20 wild type cats. As verified by direct DNA sequencing, both sensitivity and specificity of this tri-primer ARMS PCR were 100%. As the multiplex ARMS PCR test can be performed in a single PCR reaction without other post-PCR procedures, it is a simple and accurate method for molecular studies of feline ADPKD.

Simple and Sensitive Method for Identification of Human DNA by Allele‐Specific Polymerase Chain Reaction of FOXP2

The forkhead box P2 (FOXP2) gene is specifically involved in speech and language development in humans. The sequence is well conserved among many vertebrate species but has accumulated amino acid changes in the human lineage. The aim of this study was to develop a simple method to discriminate between human and nonhuman vertebrate DNA in forensic specimens by amplification of a human-specific genomic region. In the present study, we designed an allele-specific polymerase chain reaction (PCR) using primers to amplify smaller than 70-bp regions of FOXP2 to identify DNA as being of human or nonhuman, including ape, origin. PCR amplification was also successfully performed using fluorescence-labeled primers, and this method allows a single PCR reaction with a genomic DNA sample as small as 0.01 ng. This system also identified the presence of human DNA in two blood stains stored for 20 and 38 years. The results suggested the potential usefulness of FOXP2 as an identifier of human DNA in forensic samples.

Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates

Saturation mutagenesis constitutes a powerful method in the directed evolution of enzymes. Traditional protocols of whole plasmid amplification such as Stratagene’s QuikChange™ sometimes fail when the templates are difficult to amplify. In order to overcome such restrictions, we have devised a simple two-primer, two-stage polymerase chain reaction (PCR) method which constitutes an improvement over existing protocols. In the first stage of the PCR, both the mutagenic primer and the antiprimer that are not complementary anneal to the template. In the second stage, the amplified sequence is used as a megaprimer. Sites composed of one or more residues can be randomized in a single PCR reaction, irrespective of their location in the gene sequence.The method has been applied to several enzymes successfully, including P450-BM3 from Bacillus megaterium, the lipases from Pseudomonas aeruginosa and Candida antarctica and the epoxide hydrolase from Aspergillus niger. Here, we show that megaprimer size as well as the direction and design of the antiprimer are determining factors in the amplification of the plasmid. Comparison of the results with the performances of previous protocols reveals the efficiency of the improved method.

Dual-probe real-time PCR assay for detection of variola or other orthopoxviruses with dried reagents

A real-time, multiplexed polymerase chain reaction (PCR) assay based on dried PCR reagents was developed. Only variola virus could be specifically detected by a FAM (6-carboxyfluorescein)-labeled probe while camelpox, cowpox, monkeypox and vaccinia viruses could be detected by a TET (6-carboxytetramethylrhodamine)-labeled probe in a single PCR reaction. Approximately 25 copies of cloned variola virus DNA and 50 copies of genomic orthopoxviruses DNA could be detected with high reproducibility. The assay exhibited a dynamic range of seven orders of magnitude with a correlation coefficient value greater than 0.97. The sensitivity and specificity of the assay, as determined from 100 samples that contained nucleic acids from a multitude of bacterial and viral species were 96% and 98%, respectively. The limit of detection, sensitivity and specificity of the assay were comparable to standard real-time PCR assays with wet reagents. Employing a multiplexed format in this assay allows simultaneous discrimination of the variola virus from other closely related orthopoxviruses. Furthermore, the implementation of dried reagents in real-time PCR assays is an important step towards simplifying such assays and allowing their use in areas where cold storage is not easily accessible.

A highly accurate, single PCR reaction for parentage assignment in Senegal sole based on eight informative microsatellite loci

From a battery of microsatellite markers (100 loci), recently identified by our group, we have selected eight for parentage assignment in Senegal sole (Solea senegalensis). This tool is based on microsatellite loci obtained from four genomic DNA libraries and one cDNA library. Within the eight loci (six from anonymous genomic DNA sequences and two located in expressed sequence tags of known genes), we have found, in an analysis of a reproductive broodstock, between nine and 16 alleles. The expected heterozygosity was between 0.616 and 0.860. In addition, we have optimized the polymerase chain reaction (PCR) conditions to amplify all loci simultaneously in a single multiplex PCR reaction, and we have tested three lots of male and female (five to six individuals) and three offspring (50–60 larvae each). The use of the eight microsatellite loci, the possibility of amplifying them in a single PCR reaction and the high value of the exclusion probability (0.9992) make this multiplex PCR method a unique tool for parentage assignment. Finally, analysing one meiotic gynogenetic progeny, we have determined the relative distance of six of these loci to the centromere, and we have also found that all of them are unlinked. All these characteristics confer this tool with a high accuracy for parentage studies and genetic population analyses of Senegal sole.

Modified allele‐specific primer–polymerase chain reaction method for analysis of susceptibility of Helicobacter pylori strains to clarithromycin

Most clarithromycin-resistant strains of Helicobacter pylori have a mutation from adenine (A) to guanine (G) at position 2142 or 2143 of the 23S rRNA gene. Our aim in this study was to develop a polymerase chain reaction (PCR)-based assay that could determine these mutations in a single reaction tube. We designed the forward primer FP2143G and the reverse primer RP2142G, which specifically anneal with the 2143G- and 2142G-mutated sequences, respectively, of the 23S rRNA gene of H. pylori. We also designed the forward primer FP-1 and reverse primer RP-1 upstream and downstream from the positions 2142 and 2143, respectively, to distinguish the wild-type A2142G and A2143G mutations from each other by amplicon sizes. DNA was extracted from 292 gastric tissue samples positive for rapid urease test, and the DNA underwent the PCR reaction. The results were compared with minimum inhibitory concentrations (MIC) for clarithromycin. Helicobacter pylori strains with A2142G, A2143G and wild type could be distinguished by amplicon sizes by a single PCR reaction. The genotyping results were correlated well with the MIC values for clarithromycin. The median MIC for clarithromycin of the wild-type strains was <0.015 microg/mL. Those of strains with 2142G or 2143G were > or =1.0 microg/mL. Our new PCR-based assay for 23S rRNA mutations of H. pylori is a useful method for detecting clarithromycin-resistant strains of H. pylori easily.

Apolipoprotein E genotyping by multiplex tetra-primer amplification refractory mutation system PCR in single reaction tube

Apolipoprotein E (APOE) plays a critical role in lipoprotein metabolism by binding to both low-density lipoprotein and APOE receptors. The APOE gene has three allelic forms, epsilon2, epsilon3, and epsilon4, which encode different isoforms of the APOE protein. In this study, we have developed a new genotyping method for APOE. Our multiplex tetra-primer amplification refractory mutation system (multiplex T-ARMS) polymerase chain reaction (PCR) was performed in a single reaction tube with six primers consisting of two common primers and two specific primers for each of two single nucleotide polymorphism (SNP) sites. We obtained definitive electropherograms that showed three (epsilon2/epsilon2, epsilon3/epsilon3, and epsilon4/epsilon4), four (epsilon2/epsilon3 and epsilon3/epsilon4), and five (epsilon2/epsilon4) amplicons by multiplex T-ARMS PCR in a single reaction tube. Multiplex T-ARMS PCR for APOE genotyping is a simple and accurate method that requires only a single PCR reaction, without any another treatments or expensive instrumentation, to simultaneously identify two sites of single nucleotide polymorphisms.

Nested multiplex PCR—A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distinguished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the cornerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detection of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reaction (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5' end of the large ribosomal subunit were used in the experiment for testing their specificity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cultures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhiza fungal species in a same plant root system.