Monthly distribution of non-EV71 and non-CVA16 enterovirus serotypes during 2009 to June 2013 in Jinan, China.

<p>Monthly distribution of non-EV71 and non-CVA16 enterovirus serotypes during 2009 to June 2013 in Jinan, China.</p

Monthly distribution of CVA6 associated with HFMD during 2009 to June 2013 in Jinan, China.

<p>Monthly distribution of CVA6 associated with HFMD during 2009 to June 2013 in Jinan, China.</p

Phylogenetic analysis based on the 5’-UTR region of HFMD cases during 2009 to June 2013 in Jinan, China.

<p>A total of 29 representative sequences (representing 12 serotypes) from this study and 12 reference strains worldwide were used to build the tree. Jinan sequences were labeled by black solid dot. Abbreviations: JN, Jinan; CHN, China.</p

The enterovirus serotypes identified in hand-foot-mouth disease during 2009 to June 2013, in Jinan, China.

<p>The enterovirus serotypes identified in hand-foot-mouth disease during 2009 to June 2013, in Jinan, China.</p

Monthly distribution of CVA10 associated with HFMD during 2009 to June 2013 in Jinan, China.

<p>Monthly distribution of CVA10 associated with HFMD during 2009 to June 2013 in Jinan, China.</p

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

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

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

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

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