•Droplet digital PCR (ddPCR) has been used for precise and sensitive quantification of virus loads.•We used ddPCR to measure IHNV loads in fish tissue samples without the need of standard curves.•We ...compared the results of ddPCR with those from a published real-time PCR assay.•The study presented here is the first report of a RT-ddPCR assay for the detection of IHNV RNA.
Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/μl while the LOD for the RT-qPCR was 0.2 PFU/μl. Good agreement (69.4–100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.
Prokaryotic expression of recombinant nucleoprotein of viral hemorrhagic septicemia virus (VHSV) Du Juan, Huazhong Agricultural University, Wuhan (China), College of Fisheries; Lan Wensheng, Shenzhen Key Laboratory of Exotic Pests, Shenzhen (China); Liu Hong, Shenzhen Key Laboratory of Exotic Pests, Shenzhen (China)
Huazhong nongye daxue xuebao,
Jun. 2012, Letnik:
31, Številka:
3
Journal Article
通过逆转录聚合酶链式反应(RT-PCR)获得编码病毒性出血性败血症病毒(Viral hemorrhagic septicemia virus, VHSV)核蛋白基因,将其克隆至原核表达载体pET28a, 并在E.coli BL21(DE3)中得到表达,用SDS-PAGE与Western-blot对表达产物进行鉴定。结果表明, 通过RT-PCR扩增获得长度为1 ...221bp核蛋白基因片段,诱导表达重组质粒pET28a-N, 经SDS-PAGE检测,IPTG终浓度为1mmol/L时, 诱导5h蛋白表达量最高, 获得的目的蛋白大小与N蛋白的预测分子质量一致,约为48ku。诱导后的菌液进行超声波破碎后, 将沉淀和上清分别用于SDS-PAGE电泳, 结果表明目的蛋白主要以包涵体的形式存在。表达蛋白经过复性、纯化,得到了较高纯度的可溶性蛋白。经Western-blot检测表明, 该表达产物能被羊抗VHSV阳性血清特异性识别。
Viral hemorrhagic septicemia virus(VHSV), the etiologic agent of viral hemorrhagic septicemia in salmonid, caused great loss in aquaculture industry. Nucleoprotein is an important protein for activation of host immunological reaction. The nucleoprotein gene of VHSV was amplified by reverse transcription-polymerase chain reaction(RT-PCR), and cloned into pET28a vector. The recombinant E.coli BL21(DE3) containing pET28a-N was induced and the expressed protein was detected by SDS-PAGE and Western-blot. The nucleoprotein gene fragment was 1 221 bp in length. The 48 ku target protein was expressed successfully
Expression and purification of acetylcholinesterase from culex by Bac-to-Bac baculovirus system and the activity determination Tan Feng, Huazhong Agricultural University, Wuhan (China), State Key Laboratory of Agricultural Microbiology; Lan Wensheng, Chinese Academy of Sciences, Beijing (China), State Key Laboratory of Integrated Management of Pest Insects and Rodents in Agriculture, Institute of Zoology; Cui Feng, Chinese Academy of Sciences, Beijing (China), State Key Laboratory of Integrated Management of Pest Insects and Rodents in Agriculture, Institute of Zoology
Huazhong nongye daxue xuebao,
Aug. 2012, Letnik:
31, Številka:
4
Journal Article
利用Trizol法从尖音库蚊中提取总RNA，构建cDNA文库，并克隆出乙酰胆碱酯酶外显子序列；利用Bac―to-Bac杆状病毒表达系统对蚊虫乙酰胆碱酯酶进行真核表达，并利用Ni-琼脂糖对酶进行纯化。采用SDS-PAGE对纯化产物进行检测，结果表明得到了纯度较高的乙酰胆碱酯酶。参照Ellman法对酶的活性进行测定，结果显示纯酶的活力为2．219×10. 4mol／(min*g)。
The ...residues of the high toxic organophosphorous can cause chronic poisoning. At present, the detection of organophosphorous residue needs complicated operation, time consuming and high cost. Here, we harvested total RNA of mosquito by Trizol methods, constructed eDNA library, and got the exon of acetylcholinesterase. The acetylcholinesterase from culex was expressed by Bac-to-Bac system, then the enzyme was purified using Ni-sepharose. The crude and purified enzyme was tested and verified by SDS-PAGE. The enzyme activity was determined by gllman methods. It will be of great significance to biosensors in organophosphorous pesticides detection.
•This is the first study about the production and characterization of specific antibody fragments obtained by phage display technology directly against IHNV.•Six specific soluble scFv antibodies ...against IHNV were obtained.•Phage display technology is more convenient for producing monoclonal antibodies than traditional hybridoma technology.
Six single-chain fragment variable (scFv) antibodies against infectious haematopoietic necrosis virus (IHNV) were selected from an antibody phage display library by phage display technology. The soluble scFv antibodies showed a molecular weight 32kDa by Western blot. Dot blot analysis revealed that the six scFv antibodies could recognize IHNV. For enzyme linked immunosorbent assay (ELISA), four scFv antibodies (P1A4, P1A12, P1D5 and P3E2) showed cross-reactivity with spring viraemia of carp virus (SVCV). However, none of the six scFv antibodies had cross-reaction with Pike fry rhabdovirus (PFRV), Soft-shelled turtle iridovirus (STIV), viral haemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the IHNV-infected cells. These scFv antibodies will be useful in diagnostic test development and pathogenesis studies for IHNV.
•This is the first study concerning the production and characterization of specific antibody fragments obtained by phage display technology directly against SVCV.•Eight specific soluble scFv ...antibodies against SVCV were obtained.•Phage display technology is more convenient for producing monoclonal antibodies than traditional hybridoma technology.
Antibody-displaying phage library was selected after three rounds of panning against spring viraemia of carp virus (SVCV) by phage display technology. Eight positive clones which could produce soluble single-chain fragment variable (scFv) antibody induced by isopropyl-beta-d-thiogalactopyranoside (IPTG) were obtained. Dot blot results showed that the eight scFv antibodies could recognize SVCV. The soluble scFv antibodies showed a molecular weight 29kD by Western blot. All scFv antibodies could recognize SVCV proteins specifically without cross-reaction with other virus proteins by ELISA. Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the SVCV-infected cells. These scFv antibodies will be useful tools to establish immunological detection methods for SVCV.
Infectious Salmon anaemia virus (ISAV) has become a threat to the salmon industry worldwide and has caused considerable economic loss. In the present study, 9 suspect cases of ISAV infection were ...identified from iced Atlantic salmons imported from Norway in 2014 through Shenzhen port (Shenzhen, China) using methods recommended by the World Organization for Animal Health. However, the results of virus isolation were negative., Based on the sequence analysis of ISAV segment 6, the 9 ISAV isolates belonged to the HPRO type, had high homology (98.3%~100.0%) and closest relationship with Norway strains. We identified the 9 positive HPRO ISAVs from 491 iced Atlantic salmons (1. 8%). Therefore, we should strengthen the quarantine of iced Atlantic salmons from Norway in case of HPRO ISAV into China.
The temples of Angkor monuments including Angkor Thorn and Bayon in Cambodia and surrounding countries were exclusively constructed using sandstone. They are severely threatened by biodeterioration ...caused by active growth of different microorganisms on the sandstone surfaces, but knowledge on the microbial community and composition of the biofilms on the sandstone is not available from this region. This study investigated the microbial community diversity by examining the fresh and old biofilms of the biodeteriorated bas-relief wall surfaces of the Bayon Temple by analysis of 16S and 18S rRNA gene sequences. The results showed that the retrieved sequences were clustered in 11 bacterial, 11 eukaryotic and two archaeal divisions with disparate communities (Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Proteobacteria; Alveolata, Fungi, Metazoa, Viridiplantae; Crenarchaeote, and Euyarch-aeota). A comparison of the microbial communities between the fresh and old biofilms revealed that the bacterial community of old biofilm was very similar to the newly formed fresh biofilm in terms of bacterial composition, but the eukaryotic communities were distinctly different between these two. This information has important implications for understanding the formation process and development of the microbial diversity on the sandstone surfaces, and furthermore to the relationship between the extent of biodeterioration and succession of microbial communities on sandstone in tropic region.
We have developed a novel optical biosensor device using recombinant methyl parathion hydrolase (MPH) enzyme immobilized on agarose by metal-chelate affinity to detect organophosphorus (OP) compounds ...with a nitrophenyl group. The biosensor principle is based on the optical measurement of the product of OP catalysis by MPH (p-nitrophenol). Briefly, MPH containing six sequential histidines (6 × His tag) at its N-terminal was bound to nitrilotriacetic acid (NTA) agarose with Ni ions, resulting in the flexible immobilization of the bio-reaction platform. The optical biosensing system consisted of two light-emitting diodes (LEDs) and one photodiode. The LED that emitted light at the wavelength of the maximum absorption for p-nitrophenol served as the signal light, while the other LED that showed no absorbance served as the reference light. The optical sensing system detected absorbance that was linearly correlated to methyl parathion (MP) concentration and the detection limit was estimated to be 4 μM. Sensor hysteresis was investigated and the results showed that at lower concentration range of MP the difference got from the opposite process curves was very small. With its easy immobilization of enzymes and simple design in structure, the system has the potential for development into a practical portable detector for field applications.
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