A Novel Multiplex Tetra-Primer ARMS-PCR for the Simultaneous Genotyping of Six Single Nucleotide Polymorphisms Associated with Female Cancers

<div><p>Background</p><p>The tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) is a fast and economical means of assaying SNP's, requiring only PCR amplification and subsequent electrophoresis for the determination of genotypes. To improve the throughput and efficiency of T-ARMS-PCR, we combined T-ARMS-PCR with a chimeric primer-based temperature switch PCR (TSP) strategy, and used capillary electrophoresis (CE) for amplicon separation and identification. We assessed this process in the simultaneous genotyping of four breast cancer–and two cervical cancer risk–related SNPs.</p><p>Methods</p><p>A total of 24 T-ARMS-PCR primers, each 5′-tagged with a universal sequence and a pair of universal primers, were pooled together to amplify the 12 target alleles of 6 SNPs in 186 control female blood samples. Direct sequencing of all samples was also performed to assess the accuracy of this method.</p><p>Results</p><p>Of the 186 samples, as many as 11 amplicons can be produced in one single PCR and separated by CE. Genotyping results of the multiplex T-ARMS-PCR were in complete agreement with direct sequencing of all samples.</p><p>Conclusions</p><p>This novel multiplex T-ARMS-PCR method is the first reported method allowing one to genotype six SNPs in a single reaction with no post-PCR treatment other than electrophoresis. This method is reliable, fast, and easy to perform.</p></div

Genotypes and allele frequencies of tested samples in this study.

a<p>increased risk allele in bold</p>b<p><i>p</i> value was calculated using chi test</p

Direct sequencing result of sample #1101–291(corresponding to the sample in lane 1, Fig.1B), which showed the combination of 5 heterogenous and 1 homogenous SNPs.

<p>Direct sequencing result of sample #1101–291(corresponding to the sample in lane 1, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062126#pone-0062126-g001" target="_blank">Fig.1B</a>), which showed the combination of 5 heterogenous and 1 homogenous SNPs.</p

Primers for the multiplex tetra-primer ARMS-PCR for SNPs associated with breast and gynecologic cancers.

a<p>Underlined is the universal tag sequence at the 5′ end of the chimeric primers.</p>b<p>Allele-specific nucleotides at the 3′ end are indicated in bold letters.</p>c<p>Specificity is increased by the introduction of a deliberate mismatch at position -1 of the polymorphism site, indicated by bold lower case letters.</p>d<p>A pair of universal primers annealing to the 5′ portion of each chimeric primer.</p

Primers used in this study.

<p>^a Position refer to the genome of XZ0906 (GenBank accession number KF746187 to KF746196).</p><p>Primers used in this study.</p

Electrophoretic migration patterns of YN12246 dsRNA.

<p>Standard discontinuous polyacrylamide gel electrophoresis was used, with a 3.5% acrylamide concentration gel and 10% acrylamide separation gel. After staining with silver nitrate, the genome of YN12246 was visualized as 10 distinct bands.</p

Phylogenetic analysis of VP1 amino acid sequences from (A) <i>Reoviridae</i> and (B) <i>Orbivirus</i> strains.

<p>(C) Phylogenetic analysis of T2 amino acid sequences from 26 <i>Orbivirus</i> strains. This analysis employed a neighbor-joining method (using the P-distance algorithm) using the MEGA software (version 5.05; <a href="http://www.megasoftware.net" target="_blank">www.megasoftware.net</a>). Bootstrap probabilities for each node were calculated using 1000 replicates. Scale bars indicate the number of amino acid substitutions per site. In Fig 4C, because many of the available sequences are incomplete, the analysis was based on partial sequences (residues 356–567 relative to the BTV-1A sequence). Abbreviations and serotypes (or strain name) are shown in the radial tree image of Fig 4. GenBank accession numbers and further details of the sequences can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136257#pone.0136257.s001" target="_blank">S1 Table</a> (supplementary data).</p

Isolation of <i>Tibet orbivirus</i>, TIBOV, from <i>Culicoides</i> Collected in Yunnan, China

<div><p>We isolated a novel virus strain (YN12246) from <i>Culicoides spp</i>. specimens collected at the China-Laos-Myanmar border in southern Yunnan Province. This virus had a cytopathic effect (CPE) on both insect cells (C6/36) and mammalian cells (BHK-21). Electron microscopy revealed the structure of the virions to be spherical with a diameter of 75 nm. Polyacrylamide gel analysis demonstrated that the viral genome consisted of 10 segments of double-stranded RNA (dsRNA), with a distribution pattern of 3-3-3-1. The coding sequences of 9 genome segments of YN12246 (Seg1, Seg3-Seg10) were obtained by high-throughput sequencing and Sanger sequencing. Comparisons of conserved genome segments 1 and 3 (Seg1 and Seg3), encoding the polymerase-VP1 and sub-core T2 protein, respectively, showed that YN12246 groups with the <i>Culicoides</i>-borne orbiviruses. The highest levels of sequence identity were detected between YN12246 and <i>Tibet orbivirus</i> (TIBOV), indicating that they belong to the same virus species (with amino acid identity of 98.8% and 96.4% for the polymerase and T2 protein, respectively). The data presented here confirm that YN12246 is a member of the TIBOV species, which was first isolated from mosquitoes in 2009. This is the first report of the isolation of TIBOV from <i>Culicoides</i>.</p></div

Scanned images of hybridization.

<p>(A) Hybridization profile of the influenza type B virus (Upper arrow). Tile region for matrix, (<i>lower</i> arrow) noninterfering hybridization profiles of RV and EV. (B) Hybridization profiles of HBoV and CoV-HKU1 (Upper arrow). Tile regions for VP1 and NP1 of HBoV, (<i>lower</i> arrow) tile regions for NP and spike of CoV-HKU1.</p

Development of a New Resequencing Pathogen Microarray Based Assay for Detection of Broad-Spectrum Respiratory Tract Viruses in Patients with Community-Acquired Pneumonia

<div><p>A Resequencing Pathogen Microarray (RPM) is a single, highly multiplexed assay for detecting and differentiating similarly related pathogens by using closely overlapping probe sets to determine a target organism’s nucleotide sequence. In this study, a new RPM (RPM-IVDC1) that consisted of 224-bp detector tiles corresponding to 9 influenza A subtypes, 11 rhinoviruses, 28 enteroviruses and 38 other respiratory viruses was developed and optimized to provide individual and simultaneous detection sensitivities ranging from 15 to 750 genomic copies for 16 common respiratory pathogens. A total of 110 consecutive patients with community-acquired pneumonia (CAP) admitted to 5 district general hospitals in Beijing during a 1-year period were assessed using the new assay. Among the children (under age 5) and adult patients (above age 18), respiratory syncytial virus (RSV) and rhinovirus (RV) were the most common etiological agents, respectively, which is consistent with reference assays. Atypical pathogens that may cause CAP-like illness, including rubella virus, measles virus, influenza type C virus, human herpesvirus (HHV) were also detected. The results show the capability of RPM-IVDC1 for the accurate detection and identification of multiple virus types, which may be of significant use in epidemic surveillance and outbreak investigations of atypical pathogens.</p> </div