Multiplex PCR Assay For Identification Research Articles

Multiplex PCR Assay for Identification of Brucella Strains including the vaccinal Strains and its differentiation from Yersinia O: 9

Multiplex PCR Assay for Identification of Brucella Strains including the vaccinal Strains and its differentiation from Yersinia O: 9

Multiplex PCR assay for identification and quantification of bovine and equine in minced meats using novel specific nuclear DNA sequences

The 2013 horsemeat scandal demonstrated the importance and significance of developing analytical methods to accurately identify and quantify bovine and equine components in meats. In this study, we successfully screened the bovine- and equine-specific nuclear DNA sequences for detection of bovine and equine fractions in minced meats, respectively. Primers and probes designed from bovine- and equine-specific nuclear DNA sequences were critically analysed against 53 species, and then their specificity was evaluated against 23 species through PCR. The conservation of primers and probes binding regions was tested in 44 different individuals of bovine or equine. The limit of detection (LOD) is 0.05 ng in both conventional and real-time PCR, and the limit of quantification (LOQ) is down to 5%. Real-time PCR confirmed that bovine- and equine-specific nuclear DNA sequences are present as a fixed copy number in the genome. A multiplex conventional PCR method was established to effectively discriminate bovine (223 bp), equine (197 bp) and reference (129 bp) in one reaction. A multiplex real-time PCR was developed and confirmed in five binary mixtures of cattle and horse DNA, exhibiting adequate parameters of trueness (R.E. ≤ 9.35%) and precision (R.S.D. ≤ 8.88%). Furthermore, we confirmed that our method held good potential in the detection of meat products in real life. The method and strategy developed in this study will play an important role in combating adulteration and maintaining a fair market.

Haplotype-specific single-locus multiplex PCR assay for identification of Pacific and Atlantic white shrimps, exotic Litopenaeus vannamei and native Litopenaeus schmitti, along the coast of Brazil

This work describes a haplotype-specific single-locus multiplex PCR assay based on 16S mitochondrial gene polymorphisms for the identification of two Litopenaeus species present along the Brazilian coast: L. schmitti, native species from this genus in the area, and L. vannamei, exotic cultivated species that can be found along many sites along Brazil. Twenty-two 16S sequences of both species were used for the design of specific primers for each species. Two amplification patterns of the 16S gene are produced, one for each species, both with the amplification of two fragments: for L. schmitti, one universal 561 bp fragment and one specific 343 bp fragment are amplified; While for L. vannamei a universal 561 bp fragment and another species-specific fragment 408 bp is produced. For validation of the method we undertook a blind test where genetic profiles were analyzed by four different people with 100% accurate ratio. This system generates unequivocal genetic profiles to discriminate the two species of Litopenaeus in 2% agarose gels. The developed method is a useful identification tool for taxonomic purposes, in the control of the illegal trade of the native species in closed periods, as well as an instrument for monitoring the presence and spread of the exotic species in the natural environment coming from occasional escapes from shrimp farms. It provides an accurate system for direct identification of the two Litopenaeus species currently found on the Brazilian coast and also in other regions of the Atlantic where the two species co-exist.

Novel multiplex PCR assay for identification and subtyping of enteroinvasive Escherichia coli and differentiation from Shigella based on target genes selected by comparative genomics.

Both Shigella and enteroinvasive Escherichia coli (EIEC) can cause enterocolitis, but they have a distinct epidemiology and public health relevance. Current culture-independent testing (CIT) methods to identify Shigella in faecal samples rely on the ipaH gene as the target, which is also found in EIEC genomes. The aim of this study was to design an assay that can identify EIEC in cultures from CIT ipaH-positive samples. Shigella and EIEC genomes were screened to find unique regions present in EIEC genomes using a comparative genomics approach and differentiating genetic loci that are suitable PCR targets were identified. The primers for these loci were designed and tested in 6501 and 104 genomes of Shigella and EIEC, respectively. An assay with two sets of multiplex PCR reactions that differentiates Shigella and EIEC based on the presence/absence of at least two out of six loci was developed and evaluated. The majority of Shigella genomes lacked all six loci, while at least two loci were present in most EIEC genomes. This assay successfully differentiated clinical EIEC from Shigella with a limit of detection of 105 cells ml-1. The sensitivity and specificity were over 95 and 99%, respectively. The assay can further subtype EIEC genomes into their genetic lineages. This new highly specific assay can assist in the identification of EIEC in ipaH PCR-positive samples and augment the public health laboratory surveillance of EIEC and shigellosis.

Characterization and diagnostic application of genomic NPM-ALK fusion sequences in anaplastic large-cell lymphoma.

Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion genes resulting from the translocation t(2;5)(p23;q35) are present in almost 90% of childhood ALK-positive anaplastic large-cell lymphomas (ALCL). Detection and quantification of minimal disseminated disease (MDD) by measuring NPM-ALK fusion transcript levels in the blood provide independent prognostic parameters. Characterization of the genomic breakpoints provides insights into the pathogenesis of the translocation and allows for DNA-based minimal disease monitoring.We designed a nested multiplex PCR assay for identification and characterization of genomic NPM-ALK fusion sequences in 45 pediatric ALCL-patients, and used the sequences for quantitative MDD monitoring. Breakpoint analysis indicates the involvement of inaccurate non-homologous end joining repair mechanisms in the formation of NPM-ALK fusions. Parallel quantification of RNA and DNA levels in the cellular fraction of 45 blood samples from eight patients with NPM-ALK-positive ALCL correlated, as did cell-free circulating NPM-ALK DNA copies in the plasma fraction of 37 blood samples. With genomic NPM-ALK fusion sequence quantification, plasma samples of ALCL patients become an additional source for MRD-assessment. Parallel quantification of NPM-ALK transcripts and fusion genes in ALCL cell lines treated with the ALK kinase inhibitor crizotinib illustrates the potential value of supplementary DNA-based quantification in particular clinical settings.

A multiplex PCR for rapid identification of Brassica species in the triangle of U

BackgroundWithin the Brassicaceae, six species from the genus Brassica are widely cultivated throughout the world as oilseed, condiment, fodder or vegetable crops. The genetic relationships among the six Brassica species are described by U’s triangle model. Extensive shared traits and diverse morphotypes among Brassica species make identification and classification based on phenotypic data alone challenging and unreliable, especially when dealing with large germplasm collections. Consequently, a major issue for genebank collections is ensuring the correct identification of species. Molecular genotyping based on simple sequence repeat (SSR) marker sequencing or the Illumina Infinium Brassica napus 60K single nucleotide polymorphism (SNP) array has been used to identify species and assess genetic diversity of Brassica collections. However, these methods are technically challenging, expensive and time-consuming, making them unsuitable for routine or rapid screening of Brassica accessions for germplasm management. A cheaper, faster and simpler method for Brassica species identification is described here.ResultsA multiplex polymerase chain reaction (MPCR) consisting of new and existing primers specific to the Brassica A, B and C genomes was able to reliably distinguish all six Brassica species in the triangle of U with 16 control samples of known species identity. Further validation against 120 Brassica accessions previously genotyped showed that the MPCR is highly accurate and comparable to more advanced techniques such as SSR marker sequencing or the Illumina Infinium B. napus 60K SNP array. In addition, the MPCR was sensitive enough to detect seed contaminations in pooled seed samples of Brassica accessions.ConclusionA cheap and fast multiplex PCR assay for identification of Brassica species in the triangle of U was developed and validated in this study. The MPCR assay can be readily implemented in any basic molecular laboratory and should prove useful for the management of Brassica germplasm collections in genebanks.

Detection of eaeA, hlyA, stx1 and stx2 genes in pathogenic Escherichia coli isolated from broilers affected with colibacillosis

Background: Foodborne outbreaks associated with shiga toxin-producing Escherichia coli (STEC) have been well documented worldwide. STECs are major causative agents of gastroenteritis in humans that may be complicated by hemorrhagic colitis (HC), hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenia purpura (TTP). OBJECTIVES: The aim of this study was to investigate the presence of virulence-associated genes including eaeA, hlyA, stx1 and stx2 in Escherichia coli strains isolated from broiler’s Colibacillosis in the northeast of Iran. METHODS: From a total of 78 E.coli strains isolated from yolk sac infection, hepatitis and pericarditis, that were referred to educational veterinary clinic during 2011-2014, subculturing of the isolates was performed using selective media and a typical colony from each sample was subjected to multiplex PCR assay for identification of the presence of STEC important virulence-associated genes (eaeA, hlyA,stx1 and stx2) causing shiga toxin-mediated diseases. RESULTS: Of 78 E.coli isolates, one isolate was positive for both eaeA and hlyA genes while negative for stx1 and stx2 genes. CONCLUSIONS: The results showed low prevalence of STEC virulence genes associated with human infections in avian pathogenic E.coli (APEC) strains isolated from different flocks of broilers affected with colibacillosis.

Development of Multiplex PCR Assay for Identification of Eight Species from Meats in Korea

Development of Multiplex PCR Assay for Identification of Eight Species from Meats in Korea

Multiplex PCR assay for identification of commonly used disarmed Agrobacterium tumefaciens strains.

The success of Agrobacterium mediated plant transformation depends to a certain extent on appropriate selection of the A. tumefaciens strain for a particular plant species. Many stages in a plant transformation procedure are prone to bacterial contamination with similar antibiotic resistance that may compromise the identity of the A. tumefaciens strain used, in turn adversely affecting success of a transformation experiment. Different primer sets were designed to exploit genetic differences among different strains of A. tumefaciens which are commonly used for plant genetic transformation, to identity confirmation as well as to distinguish them from one another. The primer sets Ach5FtsZ-F/R specific for Ach5 and C58GlyA-F/R specific for C58 were designed on chromosomal DNA while primer sets pTiBo542-F/R and nptI-F/R specific for plasmid pTiBo542 are capable to identify and distinguish these strains from one another. These primer sets when used simultaneously in multiplex PCR, produce a pattern which uniquely identifies all these strains and distinguishes them except for GV3101 and C58C1, which can further be distinguished from each other by rifampicin screening. The multiplex PCR assay and primers being reported here serve as a valuable tool in determining the identity of A. tumefaciens strains at any stage of plant transformation procedure.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-358) contains supplementary material, which is available to authorized users.

Development and validation of a multiplex PCR assay for identification of the epidemic ST-258/512 KPC-producing Klebsiella pneumoniae clone

The Klebsiella pneumoniae carbapenemase–producing Klebsiella pneumoniae (KPC-KP) sequence type (ST)-258/512 clone is the dominant clone by which KPC has disseminated worldwide. Standard typing methods are time-consuming and are therefore impractical for identification of this clone in the course of an outbreak. Through comparative genomic study, we have previously identified several presumably unique genes of this clone: 1) PILV-like protein (pilv-l), 2) transposase, IS66-family (is-66), and a 3) phage-related protein (prp). Our aims were to 1) test for the presence of these genes using a multiplex PCR in a large, multinational collection of KPC-KP isolates and to 2) validate this assay as a typing method for the identification of the ST-258/512 clone. KPC-KP isolates (n=160) that included both ST-258/512 (group A, n=114) and non–ST-258 (group B, n=46) strains were collected from the following countries: Greece, 20; Israel, 93; Italy, 19; USA, 25; and Colombia, 3. Group B included 30 different STs from various lineages. The pilv-l gene was present in 111/114 of ST-258 isolates, including all of the KPC-negative isolates resulting in a sensitivity of 97%. Using primers for a unique ST-258 pilv-l allele resulted in a specificity of 100%. The sensitivity values of is-66 and prp genes for detecting KPC-KP ST-258 were 83 and 89%, respectively, and the specificity values were 67 and 93%, respectively. PCR for the unique pilv-l ST-258 allele provides a reliable tool for rapid detection of the ST-258 clone. This method can be helpful both in the setting of an outbreak and in a large-scale survey of KPC-KP strains.

Refining the pathovar paradigm via phylogenomics of the attaching and effacing Escherichia coli

The attaching and effacing Escherichia coli (AEEC) are characterized by the presence of a type III secretion system encoded by the locus of enterocyte effacement (LEE). Enterohemorrhagic E. coli (EHEC) are often identified as isolates that are LEE+ and carry the Shiga toxin (stx)-encoding phage, which are labeled Shiga toxin-producing E. coli; whereas enteropathogenic E. coli (EPEC) are LEE+ and often carry the EPEC adherence factor plasmid-encoded bundle-forming pilus (bfp) genes. All other LEE+/bfp-/stx- isolates have been historically designated atypical EPEC. These groups have been defined based on the presence or absence of a limited number of virulence factors, many of which are encoded on mobile elements. This study describes the comparative analysis of the genomes of 114 LEE+ E. coli isolates. Based on a whole-genome phylogeny and analysis of type III secretion system effectors, the AEEC are divided into five distinct genomic lineages. The LEE+/stx+/bfp- genomes were primarily divided into two genomic lineages, the O157/O55 EHEC1 and non-O157 EHEC2. The LEE+/bfp+/stx- AEEC isolates sequenced in this study separated into the EPEC1, EPEC2, and EPEC4 genomic lineages. A multiplex PCR assay for identification of each of these AEEC genomic lineages was developed. Of the 114 AEEC genomes analyzed, 31 LEE+ isolates were not in any of the known AEEC lineages and thus represent unclassified AEEC that in most cases are more similar to other E. coli pathovars than to text modification AEEC. Our findings demonstrate evolutionary relationships among diverse AEEC pathogens and the utility of phylogenomics for lineage-specific identification of AEEC clinical isolates.

Simple Multiplex PCR Assay for Identification of Beijing Family Mycobacterium tuberculosis Isolates with a Lineage-Specific Mutation in Rv0679c

The Beijing genotype of Mycobacterium tuberculosis is known to be a worldwide epidemic clade. It is suggested to be a possibly resistant clone against BCG vaccination and is also suggested to be highly pathogenic and prone to becoming drug resistant. Thus, monitoring the prevalence of this lineage seems to be important for the proper control of tuberculosis. The Rv0679c protein of M. tuberculosis has been predicted to be one of the outer membrane proteins and is suggested to contribute to host cell invasion. Here, we conducted a sequence analysis of the Rv0679c gene using clinical isolates and found that a single nucleotide polymorphism, C to G at position 426, can be observed only in the isolates that are identified as members of the Beijing genotype family. Here, we developed a simple multiplex PCR assay to detect this point mutation and applied it to 619 clinical isolates. The method successfully distinguished Beijing lineage clones from non-Beijing strains with 100% accuracy. This simple, quick, and cost-effective multiplex PCR assay can be used for a survey or for monitoring the prevalence of Beijing genotype M. tuberculosis strains.

Erratum: Development of a rapid multiplex PCR assay for identification of the three common Hemoglobin‐Lepore variants (Boston‐Washington, Baltimore, and Hollandia) and identification of a new Lepore variant

Erratum: Development of a rapid multiplex PCR assay for identification of the three common Hemoglobin‐Lepore variants (Boston‐Washington, Baltimore, and Hollandia) and identification of a new Lepore variant

Development of a rapid multiplex PCR assay for identification of the three common Hemoglobin‐Lepore variants (Boston‐Washington, Baltimore, and Hollandia) and identification of a new Lepore variant

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Multiplex PCR assay for rapid identification of three endangered snake species of India

Species identification has been the core issue in all approaches of conservation of endangered wild life. In this regard molecular techniques for species authentication have proved indispensable. A novel multiplex PCR assay for the identification of three Indian snake species Python morulus, Ptyas mucosus, and Naja naja is successfully demonstrated using 16S rRNA gene. Three reverse primers and a common forward primer were designed to generate three different size species-specific PCR fragments. Absence of any PCR amplification in non-target species proves the specificity of the primers. These four primers were combined in a multiplex assay to enable identification of three snake species in a single reaction. The assay described here shows its utility in identifying unknown snake specimen and in case of samples yielding low quality DNA. This multiplex PCR technique using novel primers is an unprecedented approach offered for forensic identification of exhibits originating from three Indian snake species. It is expected that this endeavor will help strengthening conservation efforts for these species.

Multiplex PCR assay for identification of Corynebacterium pseudotuberculosis from pure cultures and for rapid detection of this pathogen in clinical samples

Corynebacterium pseudotuberculosis is the aetiological agent of caseous lymphadenitis (CLA), a debilitating disease of sheep and goats. Accurate diagnosis of CLA primarily relies on microbiological examination, followed by biochemical identification of isolates. In an effort to facilitate C. pseudotuberculosis detection, a multiplex PCR (mPCR) assay was developed targeting three genes of this bacterium: the 16S rRNA gene, rpoB and pld. This method allowed efficient identification of 40 isolates of this bacterium that had been identified previously by biochemical testing. Analysis of taxonomically related species did not generate the C. pseudotuberculosis mPCR amplification profile, thereby demonstrating the assay's specificity. As little as 1 pg of C. pseudotuberculosis genomic DNA was detected by this mPCR assay, demonstrating the sensitivity of the method. The detection limit in clinical samples was estimated to be 10(3) c.f.u. C. pseudotuberculosis could be detected directly in pus samples from infected sheep and goats (n=56) with a high diagnostic sensitivity (94.6 %). The developed assay significantly improves rapid C. pseudotuberculosis detection and could supersede bacteriological culture for microbiological and epidemiological diagnosis of CLA.