Screening of DNA Barcode of Quarantine Phytophthora

Phytophthora is genus of plant-damaging Oomycetes, whose member species are capable of causing enormous economic losses on crops worldwide. In the present study, four candidate genes ITS, CO1, EF-1α and β-tubulin were tested using 123 strains of 80 species of Phytophthora to investigate the feasibility of ser- ving as DNA barcoding markers. The results showed that among the four candidate genes, ITS and CO1 had the highest success rate of PCR amplification and se- quencing, up to 100% and 96.7%. There were obvious barcode gaps in ITS, CO1 andβ-tubulin, but their frequency distributions of intra- and interspecific genetic distances were slightly overlapped. Wilcoxon rank sum test on intraspecific genetic distances of the four genes showed ITS = CO1 = β - tubulin = EF-1α indicating they bad the same effect on intraspecific discrimination, while the test on interspecific genetic distances of the four genes showed ITS 〉 C01 〉 β- tubulin 〉 EF - 1α. In summary, ITS and COl should be used in combination as the primary barcodes, β-tubulin as the complementary barcede for the identification of 11 quaran- tine Phytophthora species.

Subtyping Animal Influenza Virus with General Multiplex RT-PCR and Liquichip High Throughput (GMPLex)

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10-5(= 280ELD50) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10-4(=2800 ELD50). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.

Development of a Liquid Chip Technique to Simultaneously Detect Taura Syndrome Virus( TSV) and Yellow Head Disease Virus( YHDV)

The aim is to develop a liquid chip technique to detect Taura syndrome virus( TSV) and yellow head disease virus( YHDV) on Penaeus orientalis simultaneously. The CP2 gene of TSV and N gene of YHDV in Gen Bank was analysed by using the software DNAStar 7. 0 to design the TSV-and YHDV-specific primers. The primers were labeled with biotin and subjected to amination modification. They were then coupled with fluorescence-coded microspheres and then used for hybridization with RT- PCR products of TSV and YHDV. The liquid chip detection technique for detection of TSV and YHDV was established by using BD FACSArray to detect fluorescence signal in the reaction system. This assay system had a high sensitivity to TSV and YHDV,with the detection of limit of 100 pg. Moreover,the assay was specific for the detection of TSV,YHDV and was not susceptible to cross with other viruses,including white spot syndrome virus( WSSV),spring viremia of carp virus( SVCV),infectious haematopoietic necrosis virus( IHNV). In conclusion,the liquid chip assay technique established in this study is highly sensitive and specific to TSV and YHDV detection. Moreover,it provides a novel,convenient and rapid approach for the detection of TSV and YHDV.

Simultaneous Determination of Ultraviolet Absorbers and Antibacterial Agents in Textiles by Ultra-High Performance Liquid Chromatography/Orbitrap High Resolution Mass Spectrometry

This paper reported a new analytical method for the simultaneous determination of seven benzotriazole ultraviolet absorbers and seven antibacterial agents in textiles. After ultrasonic extraction for the textile samples in methanol, the solutions were analyzed by ultra-high performance liquid chromotagraphy/orbitrap high resolution mass spectrometry (UPLC/Orbitrap HRMS). It showed that a good chromatographic separation for these target compounds was achieved by a Hypersil GOLD column (100 mm × 2.1 mm × 1.9 μm) with a gradient elution of methanol and 0.1% aqueous formic acid solution (containing 0.5 mmol/L ammonium acetate). Triclosan and 4-chloro-3,5-dimethyl phenol (PCMX) were detected by the orbitrap HRMS in an electrospray ionization (ESI) negative mode while the other twelve target compounds were detected by orbitrap HRMS in ESI positive mode. Full scan experiment was performed over the range from m/z 100 to m/z 500. These target compounds were routinely detected with mass accuracy below 2 × 10-6 (2 ppm) at the optimized conditions. The results showed that the limits of detection (LODs) were in the range from 0.1 to 0.3 μg/kg. The blank samples were spiked at three levels and their average recoveries varied from 80.5% to 96.3% while the relative standard deviation (RSD) changed from 3.2% to 9.9%. The present method was also applied for the determination of those ultraviolet absorbers and antibacterial agents in the commercial textiles.

Isolation, identification and genomic characterization of the Asian lineage Zika virus imported to China

Dear Editor,Zika virus(ZIKV)used to be an unknown mosquito-borne flavivirus,and maintained its limited sylvatic circulation in a few African and Asian countries(Enfissi et al.,2016).Based on available clinical data,the symptoms in human infections with ZIKV are supposed to be similar to other arbovirus infections such as dengue,and characterized by fever,skin rashes,conjunctivitis,muscle and joint pain,malaise,and headache(Duffy et al.,2009).However。

Isolation and characterization of Zika virus imported to China using C6/36 mosquito cells

Dear Editor,Zika virus(ZIKV)is a mosquito-borne flavivirus that usually causes asymptomatic infections or mild illness in humans.However,the unprecedented epidemics of ZIKV in Latin America since early 2015 have made this flavivirus an international health risk(Liu and Zhang,2016).In particular,the potential association of ZIKV infection with the remarkable increase in the number

A new visual investigation into nanogold-based genechip assay by atomic force and scanning tunneling microscope

Conformations of surface atoms in various stages of nanogold-based genechip testing are scanned by the atomic force and scanning tunneling microscope. We intuitively observe the process and differences in probe combination, nucleic acid hybridization, and silver staining, which might be useful to validate the assay method of genechip. We hope to use this technology to make the other invisible chemical or biochemical reaction become visible and convincible in the future.