Development of an all-optical framing camera and its application on the Z-pinch.

An all-optical framing camera has been developed which measures the spatial profile of photons flux by utilizing a laser beam to probe the refractive index change in an indium phosphide semiconductor. This framing camera acquires two frames with the time resolution of about 1.5 ns and the inter frame separation time of about 13 ns by angularly multiplexing the probe beam on to the semiconductor. The spatial resolution of this camera has been estimated to be about 140 µm and the spectral response of this camera has also been theoretically investigated in 5 eV-100 KeV range. This camera has been applied in investigating the imploding dynamics of the molybdenum planar wire array Z-pinch on the 1-MA "QiangGuang-1" facility. This framing camera can provide an alternative scheme for high energy density physics experiments.

Development of a Liquid Chip Technique to Simultaneously Detect Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, and Viral Hemorrhagic Septicemia of Salmonids.

A liquid chip technique was developed to detect spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV) of salmonids simultaneously. Sequences of the G gene of SVCV, N gene of IHNV, and G gene of VHSV were used to design SVCV-, IHNV-, and VHSV-specific primers, which were labeled with biotin and subjected to amination modification. They were then coupled with fluorescence-coded microspheres and used for hybridization with reverse-transcription PCR products of SVCV, IHNV, and VHSV. A BD FACSArray was used to detect fluorescence signal in the reaction system. This assay system had a high sensitivity to SVCV, VHSV, and IHNV, with LODs of 10, 10, and 100 pg/µL, respectively. Moreover, the assay was specific for the detection of SVCV, IHNV, and VHSV and was not susceptible to cross-detection of other viruses, including pike fry rhabdovirus, hirame rhabdovirus, infectious pancreatic necrosis virus, viral nervous necrosis virus, yellowtail ascites virus, grass carp reovirus, red sea bream iridovirus, and koi herpesvirus. The liquid chip assay technique established in this study provides a novel, convenient, and rapid approach for the detection of SVCV, IHNV, and VHSV.

[Studies and application method and quality control system for detecting hepatitis A virus in water for fruits and vegetables production].

OBJECTIVE: To explore detecting methods and quality control standard system for hepatitis A virus( HAV) in water for fruits and vegetables production. METHODS: 100 µL of coliphage MS2( 2. 5 × 10~9 pfu / mL) was added into each water sample, and positively charged membrane filter method was used to capture virus. The virus extraction efficiency of each sample can be calculated according to standard curve of coliphage. Then the quality control system and real-time RT-PCR method were established. RESULTS: This research compared the extraction efficiency of HAV and coliphage MS2 which were added into the water samples at the same time. The extraction efficiency of HAV was from 1. 24% to 32. 68%, and coliphage MS2 1. 64% to 14. 24%. The efficiency met the requirements of ISO / TS 15216-2-2013. Meanwhile, the HAV in 30 water samples were detected, and one was positive. The extraction efficiency of coliphage MS2 was from1. 24% to 24. 19%, and the standard deviation was 0. 0612. CONCLUSION: This research establish a quality control system to ensure the quality of test results.

[Risk Assessment and Genotyping of Hepatitis A Virus in Fruit and Vegetable Products].

This study explored risk assessment and genotyping of hepatitis A virus(HAV)in fruit and vegetable products. Two hundred and sixteen samples of fruit and vegetable products were examined by real-time RT-PCR. Six samples tested positive for hepatitis A virus, including frozen strawberries, frozen blueberries, frozen diced potatoes, frozen diced apple and frozen raspberries, accounting for 2.8% of the total samples tested. These six HAV isolates were genotyped by nested RT-PCR amplification, and a single band was detected in isolates from frozen diced apple(210-1999)and frozen blueberries(210-2002).These two isolates belong to the HAV IB subtype, based on analysis of evolution and homology. This study provides HAV risk information for fruit and vegetable enterprises and food safety management departments. Furthermore, it lays a foundation for HAV traceability, and provides technical support to ensure product safety for enterprises at critical control points including planting, harvest, processing and packaging. These results provide reliable data for epidemiological diagnosis.

A sensitive and specific hyperbranched rolling circle amplification assay and test strip for white spot syndrome virus.

White spot syndrome virus (WSSV) is a global threat to the prawn industry, and there is no simple method for field-based testing of this virus. We designed a padlock probe and primers to the capsid protein gene VP28 of WSSV, and established a hyperbranched rolling circle amplification (HRCA) assay and a corresponding strip-based test. The assay and the test strip both had similar high accuracy and specificity, and their sensitivity was about 10 copies/µL, which is 100 times higher than conventional PCR. In this study, 68 batches of prawns were tested for WSSV with the HRCA assay and test strip, and the results were compared with the PCR assay. The results indicated that both the assay and test strip had accuracy similar to each other and to the PCR results. However, the assay and strip were more sensitive and user-friendly than PCR. Establishment of this method will provide a rapid detection of WSSV and also a basis for field-based detection of animal disease.

Analysis of the bacterial diversity existing on animal hide and wool: development of a preliminary PCR-restriction fragment length polymorphism fingerprint database for identifying isolates.

Twenty-one bacterial strains were isolated from imported cattle hide and rabbit wool using two types of media, nutrient broth, and nutrient broth with serum. The bacteria identified were Brevibacillus laterosporus, Leclercia adecarboxylata, Peptococcus niger, Bacillus circulans, Raoultella ornithinolytica, Bacillus subtilis, Bacillus cereus, Bacillus thermobacillus, Bacillus choshinensis, Bacillus sphaericus, Acinetobacter haemolyticus, Sphingomonas paucimobilis, Bacillus thuringiensis, Staphylococcus intermedius, Mycobacteria, Moraxella, Klebsiella pneumoniae, Ralstonia pickettii, Staphylococcus chromogenes, Comamonas testosteroni, and Cupriavidus pauculus. The 16s rDNA gene of each bacterium was amplified using the universal primers 27f and 1492r. The amplicons were digested with AvaI, BamHI, BgII, DraI, EcoRI, EcoRV, HindIII, HinfI, HpaI, PstI, SmaI, TaqII, XbaI, XmaI, AluI, XhoI, and PvuI individually. A specific fingerprint from the PCR-restriction fragment length polymorphism method based on 16s rDNA was obtained for each bacterium. The results showed that the method developed was useful not only for bacterial identification but also for the etiological investigation of pathogens in imported animal hair and wool.

Development and evaluation of a sensitive and quantitative assay for hirame rhabdovirus based on quantitative RT-PCR.

The aim of the present work was to develop a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) assay using a TaqMan probe to detect and quantify hirame rhabdovirus (HRV). The results demonstrated that the assay had a detection limit of 100 copies of RNA per reaction and a log-linear range up to 10(8) copies of HRV RNA. Regression analysis demonstrated a significant correlation with an R(2) value of 0.9963 and a slope of -3.18 between the mean C(t) values and HRV cRNA. This assay was 100 times more sensitive than the conventional one-step RT-PCR assay. The qRT-PCR assay was found to be highly reproducible with intra- and inter-assay coefficients of variation of 0.37-1.72% and 1.37-3.79%, respectively. The primers and TaqMan probe were specific for HRV and did not react with either the spring viraemia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV), marine birnavirus (MABV), viral hemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). This assay was evaluated using 40 fish samples, indicating that such method offers considerable advantages over the classical virus isolation method currently used for HRV surveillance. In conclusion, the developed qRT-PCR assay was a reliable, specific and sensitive tool for the quantitative diagnosis of HRV in fish samples.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid detection of lymphocystis disease virus.

A loop-mediated isothermal amplification (LAMP) assay was developed for the detection of lymphocystis disease virus (LCDV). A set of five specific primers, two inner and two outer primers and a loop primer, were designed on the basis of the major capsid protein gene of LCDV. The reaction time and temperatures were optimized for 60 min at 63 degrees C, respectively. LAMP amplification products were detected by a ladder-like appearance on agarose gel electrophoresis or a naked-eye inspection of a color change in the reaction tube by addition of SYBR Green I. The assay was specific for LCDV, and there was no cross-reactivity with white spot syndrome virus (WSSV) or six other Iridoviridae viruses (epizootic hematopoietic necrosis virus, EHNV; tiger frog virus, TFV; Bohle iridovirus, BIV; soft-shelled turtle iridovirus, STIV; infectious spleen and kidney necrosis virus, ISKNV; red sea bream iridovirus, RSIV). The detection limit of the LAMP assay was 15 fg, which was similar to that of real-time quantitative polymerase chain reaction (PCR) and 10-fold higher than the conventional PCR. The LAMP assay was evaluated using 109 clinical samples, and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for detection of LCDV. The LCDV LAMP assay has potential for early diagnosis of LCDV infection.

Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR.

A one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) using a TaqMan probe to quantitatively detect spring viremia of carp virus (SVCV) is described. In this assay, a pair of primers amplifying an 81-bp DNA fragment and a TaqMan probe was designed targeting the conserved region at the SVCV glycoprotein (G) gene. To avoid the disadvantages arising from plasmids, an extension adding a T7 phage polymerase promoter to the 5' end of the antisense primer was carried out to obtain viral cRNA. Standardized cycle threshold (Ct) values for 10-fold serial dilutions of SVCV cRNA were achieved by real-time RT-PCR and used to create standard curves. A regression line between the mean Ct values and viral template concentrations over a 1:10(7) dilution range with an r(2) value (0.9916) and a slope (-3.36) and the coefficient of variation (intra- or inter-assay is <2% and <4%, respectively) indicated that the assay was highly reproducible. The assay was specific to SVCV and there was no cross-reactivity with other fish viruses (viral hemorrhagic septicemia virus, VHSV; infectious pancreatic necrosis virus, IPNV; grass carp reovirus, GCRV; epizootic haematopoietic necrosis virus, EHNV). The standard curve allows precise absolute quantitation and shows that the detection limit of the assay is 40 copies of the viral RNA. This one-step RT-PCR assay was evaluated using 60 clinical common carp samples collected during the year 2006, indicating such technology offers considerable advantages over conventional RT-PCR methods in current routine use for SVCV surveillance. This is the first report of the development of a one-step TaqMan RT-PCR for SVCV detection.

Development of real-time polymerase chain reaction assay with TaqMan probe for the quantitative detection of infectious hypodermal and hematopoietic necrosis virus from shrimp.

An assay was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) based on real-time quantitative polymerase chain reaction (PCR). A pair of primers and a TaqMan probe were designed that are specific for the recognition of a conservative region in the IHHNV genome. The IHHNV real-time PCR assay had a detection limit of 9 DNA copies, with a dynamic range of detection between 9 x 106 and 9 DNA copies. The primer pairs and probe were specific to IHHNV and did not cross-react with shrimp genomic DNA or other shrimp viruses such as White Spot Syndrome Virus (WSSV), Monodon Baculovirus (MBV), and hepatopancreatic parvovirus (HPV). This assay has a broad application for basic and clinical investigations. For clinical samples, the real-time PCR assay detected all the positive samples screened by conventional PCR, which indicated the sensitivity of the real-time assay. The IHHNV real-time PCR assay with high sensitivity, specificity, wide range of detection ability, and simplicity is particularly useful for screening large numbers of specimens and measuring viral loads to monitor the broodstock.

[Current status and future perspective of genetic linkage mapping in aquaculture species].

Constructing genetic linkage map is an essential tool to acknowledge genome in aquaculture species. This paper has reviewed the current status of genetic linkage map research, including mapping population, mapping method and molecular markers used to construct linkage map. Linkage map has great potential in marker assisted selection (MAS), gene locating and cloning, and comparative genome mapping. Genetic linkage map with high density and wide coverage of genome will allow cloning the genes which contribute to economically important traits. The ultimate aim of the constructing linkage map is the development of fast-growing, disease-resistant strains of the major aquaculture species.