Development and evaluation of a direct TaqMan qPCR assay for the rapid detection of diverse carnivore amdoparvoviruses.

Amdoparvoviruses infect carnivore species, including mink, raccoon dog, fox, skunk, and red panda. Amdoparvovirus infection is a major cause of morbidity and mortality in farmed minks. Here, we developed a direct TaqMan qPCR assay for detection and quantification of carnivore amdoparvoviruses by using three primers and one probe based on the conserved VP2 gene. The detection limit for Aleutian mink disease virus (AMDV) and Raccoon dog and arctic fox amdoparvovirus (RFAV) were 4.06 × 101 copies/µl and 2.93 × 101 copies/µl, respectively. Both intra- and inter-assay variability were less than 2%. Among 74 carnivore samples, the positive rates for amdoparvoviruses were 62.2% (46/74) by direct TaqMan qPCR, while only 40.5% (30/74) by SYBR Green I qPCR. This result suggests that the direct TaqMan qPCR was more sensitive than the SYBR Green I qPCR. Additionally, the direct TaqMan qPCR is a rapid and sensitive method for liquid samples at microliter level as the assay employed the direct alkaline lysis method to obtain viral DNA and, therefore, eliminated the cumbersome steps in extracting DNA. Overall, the direct TaqMan qPCR assay possessed high specificity, sensitivity, and reproducibility, indicating that it can be used as a powerful tool for detection and quantification of various carnivore amdoparvoviruses in epidemiological and pathogenesis studies.

Fine Mapping and Candidate Gene Prediction for White Immature Fruit Skin in Cucumber (Cucumis sativus L.).

In this study, a single recessive gene (designated w0) was identified to control the white immature fruit color. Genetic mapping with simple sequence repeats (SSR) markers located the w0 gene in the distal region of cucumber chromosome 3 (Chr.3). Fine mapping was then conducted using the method of draft genome scaffold-assisted chromosome walking with 7304 F2 individuals, which allowed for the assignment of the gene locus to a 100.3 kb genomic DNA region with two flanking markers, Q138 and Q193. Thirteen candidate genes were predicted in the 100.3 kb region. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that the expression of the Csa3G904140 gene, which encodes a two-component response regulator-like protein, was much higher in the immature fruit skin of the green parental line (Q1) than in the white parental line (H4). A coding sequence analysis suggested that a single-base insertion occurred at the ninth exon, resulting in a frameshift mutation in Csa3G904140 of H4, and the mutation was consistent with the phenotype in 17 green/white germplasms. Therefore, Csa3G904140 was taken as the likely candidate gene controlling the immature fruit color of cultivated cucumber. This study will contribute to the cloning of candidate genes and the development of white cucumber cultivars using marker-assisted breeding.

[Optimization of processing technologies for dipsaci radix habitat by comprehensive evaluation method].

OBJECTIVE: To optimize the processing technologies of Dipsaci Radix by comprehensive method. METHODS: According to the Chinese Pharmacopoeia (2010 edition), UV Spectrophotometry and HPLC analysis were used to determine the contents of total saponins, saponins VI of water extract and alcohol extract of Dipsaci Radix. Comprehensive evaluation method was used to optimize the processing technologies for Dipsaci Radix habitat. RESULTS: The sequence of quality of processing was as follows: baked half dry sweating products (0.7046) > half dry sweating products (0.5857) in the shade > scald soft sweating products (0.5852) > bask dried products (0.3138) > evaporate soft sweating products (0.0952). CONCLUSION: The processing technology optimized by the comprehensive method can ensure the quality of Dipsaci Radix.

[Study on quality standards of decoction pieces of Indian stringbush root with sweat].

OBJECTIVE: To study quality standards of 10 batches of Indian of stringbush root decoction pieces with sweat processing and build up the quality standard. METHODS: 10 batches of indian stringbush root decoction pieces with sweat were investigated with TLC. Moisture content, total ash, acid-insoluble ash and extractum were explored. The content of daphnoretin was determined by HPLC. RESULTS: Indian stringbush root decoction pieces with sweat,the moisture content should not pass 14.5%, total ash should not pass 3.5%, acid-insoluble ash should not pass 1.0%, alcohol-soluble extractive should not lower than 9.0%, the content of daphnoretin should not lower than 0.2%. CONCLUSION: Quality control quantization evaluation system of Indian stringbush root decoction pieces with sweat is establishment initial.

The complete chloroplast genome of Abies georgei Orr var. smithii, a species endemic to the Qinghai-Tibet Plateau, China.

Abies georgei Orr var. smithii is an evergreen coniferous species of Pinaceae, and is endemic to the Qinghai-Tibet Plateau of China. Considering its vital ecological functions in this unique area, the complete chloroplast (cp) genome was constructed in this study to provide genetic information for its further study of conservation and evolution. The complete cp genome is 121,213 bp in length with GC content of 38.3%, and contains a tetrad structure, including a large single copy region of 76,278 bp, a small single copy of 42,575 bp, and two very short repeats of 1,180 bp for each. Besides, it contains 113 genes in total, including 74 CDSs, 35 tRNAs, and four rRNAs. This genome has been deposited in Genbank under accession number of MT527722.

[Genetic variation analysis of canine parvovirus VP2 gene in China].

To recognize the molecular biology character, phylogenetic relationship and the state quo prevalent of Canine parvovirus (CPV), Faecal samnples from pet dogs with acute enteritis in the cities of Beijing, Wuhan, and Nanjing were collected and tested for CPV by PCR and other assay between 2006 and 2008. There was no CPV to FPV (MEV) variation by PCR-RFLP analysis in all samples. The complete ORFs of VP2 genes were obtained by PCR from 15 clinical CPVs and 2 CPV vaccine strains. All amplicons were cloned and sequenced. Analysis of the VP2 sequences showed that clinical CPVs both belong to CPV-2a subtype, and could be classified into a new cluster by amino acids contrasting which contains Tyr-->Ile (324) mutation. Besides the 2 CPV vaccine strains belong to CPV-2 subtype, and both of them have scattered variation in amino acids residues of VP2 protein. Construction of the phylogenetic tree based on CPV VP2 sequence showed these 15 CPV clinical strains were in close relationship with Korea strain K001 than CPV-2a isolates in other countries at early time, It is indicated that the canine parvovirus genetic variation was associated with location and time in some degree. The survey of CPV capsid protein VP2 gene provided the useful information for the identification of CPV types and understanding of their genetic relationship.