一例猪伪狂犬病病毒的诊断

2015年11月,当地某养殖户15日龄仔猪出现角弓反张、精神沉郁等疑似猪伪狂犬病病毒感染的典型神经症状,但查看免疫程序,已免疫猪伪狂犬病疫苗（Bartha-k61株）。经gE基因血清学调查和针对gE基因的PCR诊断,证实该病例为猪伪狂犬病病毒感染所致。     

Immuno-PCR in cancer and non-cancer related diseases: a review

Polymerase chain reaction-amplified immunoassay (immuno-PCR, iPCR) is a method that combines the specificity of an immunological detection method and the sensitivity of a nucleic acid amplification method. In this way, immuno-PCR uses a minimum amount of sample, and allows the detection of rare diseases and those diseases in very early stage (i.e. infectious diseases, degenerative disorders, or neoplastic diseases). The present review was aimed to describe this new methodology and applications to the early detection of cancer and non-cancer related diseases, and discuss about the possibility to detect diverse biomarkers of oncology disorders, such as breast, gastric, colorectal and nasopharynx cancer, and other factors related to the growth of the neoplastic disease.     

Establishment and Application of Single and Duplex RT-PCR Method for Differentiation of Wild-type and Vaccine Viruses of Classical Swine Fever Virus

For the distinguishment of wild-type and vaccine viruses of classical swine fever (CSF),a rapid and simple method was established,which laid the foundation for the purification of classical swine fever virus (CSFV) in large-scale farms.It was found that T-rich insertion sequence independently that 3'-NTR in Lapinized vaccine strain according to the genome sequence comparative analysis of CSF wild virus,vaccine virus and near origin virus published in GenBank,on the basis of this,two pairs of specific primers were designed in the upper and lower ends of the insertion sequence and duplex RT-PCR primers were designed.The anneal temperature of polymerese chain reaction (PCR) was optimized,then the two single and duplex RT-PCR method for the differentiation of wild-type and vaccine viruses of CSF were established.The sensitivity and specificity results showed that the minimum amounts of nucleic acid by the single RT-PCR were 2.2 pg (one pair of primers in single RT-PCR)and 1.7 pg (another pair of primers in single RT-PCR),respectively,and that of the duplex RT-PCR were 8.2 pg (wild-type virus of CSF) and 6.7 pg (vaccine virus of CSF), respectively, and no amplification of PRV,PRRSV,JEV,BVDV,PCV2 DNA/RNA were detected by these methods.Then the methods were used to detect 146 suspicious clinical samples,and the results showed that the positive rates of the mixed infection by wild-type and vaccine viruses of CSF in breeding sows,fattening pigs,nursery pigs and suckling piglets were 6.3%,7.4%,8.3%,8.6%, respectively.The results indicated that the single and duplex RT-PCR methods had high specificity,sensitivity,and good repeatability,and it had an important reference value for the purification of CSF in large-scale farms.     

A Resazurin Microplate Assay for Quick Diagnosis and Antibiotic Sensitivity Measurement of Bovine Mastitis-causing E.coli

To establish a microplate assay for quick diagnosis and antibiotic sensitivity measurement of bovine mastitis-causing E.coli,the phenol red indicator in E.coli selective medium was replaced with resazurin and the resazurin microplate assay was established by determing of optimization of indicator concentration,bacterial inoculation dose and culture time.The testing results of different bacteria and their infection-mimicking milk samples showed that the microplate assay had a strong selective property for E.coli and the definite diagnostic result could be obtained within 10 h with a capability of reflecting infection strength of dairy cow mammary gland.The results of 89 mastitis-positive milk samples showed that the microplate assay had a detection sensitivity of 100%,and the negative milk samples specificity was 94.9%,95.5% agreement to the conventional culture-based method.The micoplates were coated with rectified concentrations of 10 different antibiotics and the quick drug sensitivity assay was performed using 10 E.coli strains,infection-mimicking milk samples and 10 E.coli-positive milk samples.The obtained results had a complete agreement with that of standard disc diffusion method.These data suggested that the resazurin microplate assay established in this study could replace the conventional methods for quick diagnosis and antibiotic sensitivity measurement of bovine mastitis-causing E.coli.     

Development and Application of Sandwich ELISA for Diagnosis of Fascioliasis gigantica in Early Stage

To establish a method for the diagnosis of Fascioliasis gigantica in early stage, five hybridoma cell lines were recovered and used for preparation of monoclonal antibodies.7D2 was used as a capture antibody and 7D1 as detection antibody.Coating antibody dilution was 1:6 400 (0.208 μg/mL), 5% nonfat milk was used as blocking solution and detection antibody dilution was 1:10 000 (0.200 μg/mL).The detection limit of the sandwich ELISA was 1:3 200 (0.156 μg/mL), with good specificity and stability, and there was no cross reaction with other kinds of parasite antigen.The results showed that the method provided the important conditions and theoretical basis for the diagnosis of Fascioliasis gigantica in early stage. This would save unnecessary economic losses to the livestock, and it had clinical application value.     

Establishment of Indirect ELISA Method for Detecting Recombinant Truncated N Protein of Porcine Epidemic Diarrhea Virus

In order to establish an indirect ELISA to detect antibody of porcine epidemic diarrhea virus (PEDV).The experiment using the recombinant and purified truncated N protein as antigen expressed in E.coli BL21(DE3),the indirect ELISA was named rnPED-ELISA.The recombinant truncated N protein antigen showed no cross-reaction with the positive sera of other 7 kinds of swine diseases,CV%of intro-batch duplicativity test and inter-batch duplicativity test were less 13%;Sensitivity and specificity of rnPED-ELISA relative to SN were 93.33% and 90.00%,respectively;rnPED-ELISA compared with TSZ PEDV antibody diagnosis Kit,91.67% concordance was obtained.200 serum samples were detected by this method,the total masculine ratio was 69.5%.Therefore,this rnPED-ELISA based on recombinant truncated N protein antigen had good sensitivity and specificity,could afforded a simple and rapidmeans for assessment of vaccination in the field and investigation of PED epidemiology.     

微生物杀虫剂联合施用对草地贪夜蛾幼虫的防治效果

为提高微生物杀虫剂对草地贪夜蛾Spodoptera frugiperda的防治效果,利用球孢白僵菌Beauveria bassiana IPPB343、IPPB1237与金龟子绿僵菌Metarhizium anisopliae IPPM330189三种昆虫病原真菌,斜纹夜蛾核型多角体病毒（Spodoptera litura nuclear polyhedrosis viruses,SLNPV）、甘蓝夜蛾核型多角体病毒（Mamestra brassicae nuclear polyhedrosis viruses,MBNPV）2种昆虫病毒和短稳杆菌Empedobacter brevis昆虫病原细菌,设置9种联合施用药剂组合,评价6种微生物杀虫剂单独使用以及不同药剂联合施用组合对草地贪夜蛾幼虫的毒力以及田间防治效果。结果表明,与6种微生物杀虫剂单独使用相比,9种联合施用组合均能提升微生物杀虫剂对草地贪夜蛾的毒力水平与田间防治效果。施药后7 d时,金龟子绿僵菌IPPM330189+MBNPV药剂组合的防治效果最好且增效最明显,对草地贪夜蛾的田间防治效果达到88.75%,协同毒力指数为23.78。此外,金龟子绿僵菌IPPM330189+短稳杆菌悬浮剂药剂组合施药后7 d对草地贪夜蛾的田间防治效果达到87.45%,协同毒力指数也可达到21.72。表明微生物杀虫剂联合施用可显著提升其对草地贪夜蛾的防治效果。     

Nipah virus: epidemiology,pathology, immunobiology and advances in diagnosis,vaccine designing and control strategies – a comprehensive review

Nipah (Nee-pa) viral disease is a zoonotic infection caused by Nipah virus (NiV), a paramyxovirus belonging to the genus Henipavirus of the family Paramyxoviridae. It is a biosafety level-4 pathogen, which is transmitted by specific types of fruit bats, mainly Pteropus spp. which are natural reservoir host. The disease was reported for the first time from the Kampung Sungai Nipah village of Malaysia in 1998. Human-to-human transmission also occurs. Outbreaks have been reported also from other countries in South and Southeast Asia. Phylogenetic analysis affirmed the circulation of two major clades of NiV as based on currently available complete N and G gene sequences. NiV isolates from Malaysia and Cambodia clustered together in NiV-MY clade, whereas isolates from Bangladesh and India clusterered within NiV-BD clade. NiV isolates from Thailand harboured mixed population of sequences. In humans, the virus is responsible for causing rapidly progressing severe illness which might be characterized by severe respiratory illness and/or deadly encephalitis. In pigs below six months of age, respiratory illness along with nervous symptoms may develop. Different types of enzyme-linked immunosorbent assays along with molecular methods based on polymerase chain reaction have been developed for diagnostic purposes. Due to the expensive nature of the antibody drugs, identification of broad-spectrum antivirals is essential along with focusing on small interfering RNAs (siRNAs). High pathogenicity of NiV in humans, and lack of vaccines or therapeutics to counter this disease have attracted attention of researchers worldwide for developing effective NiV vaccine and treatment regimens.     

口蹄疫诊断技术的研究进展

口蹄疫是一种重要的人畜共患传染病,可使世界范围内的畜牧业遭受严重的经济损失,阻碍畜牧业、畜产品加工业的发展,并危害人体健康。各国都采取了多种措施控制该病的发生。而防制口蹄疫的重要环节是要作出及时、准确的诊断。作者对口蹄疫诊断技术研究进展,包括ELISA诊断技术研究以及分子生物学诊断技术研究进展进行了综述,并对口蹄疫诊断技术进行了展望。