多重RT-PCR方法检测甲型流感病毒

目的建立一种快速、敏感、特异的多重RT-PCR,同时检测甲型流感病毒中的3个分型:甲型H1N1流感病毒,季节性H1N1流感病毒,季节性H3N2流感病毒,并将此方法应用到实验室流感病毒核酸检测技术中。方法利用甲型流感病毒3个分型病毒的引物,在同一个RT-PCR反应体系中,对疑似流感咽拭子标本进行检测。结果多重RT-PCR对甲型流感病毒中分型病毒有较高的灵敏度和特异性,可直接从疑似流感标本中同时进行甲型流感病毒分型检测。结论此实验中采用的多重RT-PCR具有与常规RT-PCR一样的特异性和敏感度,而且比普通RT-PCR和病毒分离法更快速,也更简便。     

Molecular characterization of a Class I Newcastle disease virus strain isolated from a pigeon in China

Constant monitoring is performed to elucidate the role of natural hosts in the ecology of Newcastle disease virus (NDV). In this study, an NDV strain isolated from an asymptomatic pigeon was sequenced and analysed. Results showed that the full-length genomes of this isolate were 15,198 nucleotides with the gene order of 3′-NP-P-M-F-HN-L-5′. This NDV isolate was lentogenic, with an intracerebral pathogenicity index of 0.00 and a mean time of death more than 148?h. The isolate possessed a motif of -¹¹²E-R-Q-E-R-L¹¹⁷- at the F protein cleavage site. In addition, 7 and 13 amino acid substitutions were identified in the functional domains of fusion protein (F) and haemagglutinin-neuraminidase protein (HN) proteins, respectively. Analysis of the amino acids of neutralizing epitopes of F and HN proteins showed 3 and 10 amino acid substitutions, respectively, in the isolate. Phylogenetic analysis classified the isolate into genotype Ib in Class I. This isolate shared high homologies with the NDV strains isolated from wild birds and waterfowl in southern and eastern parts of China from 2005 to 2013. To our knowledge, this study is the first to report a NDV strain isolated from pigeon that belongs to genotype Ib in Class I, rather than to the traditional genotype VI or other sub-genotypes in Class II. This study provides information to elucidate the distribution and evolution of Class I viruses for further NDV prevention.     

Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCR with nucleocapsid gene-specific primers

SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (C_t) 18.19 ± 3.63 and a melting temperature (T_m) 86.0 ± 0.28 °C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the C_t value 14.70 ± 2.32 and T_m 87.4 ± 0.21 °C. The assay had a dynamic detection range which spans over a 5 log₁₀ concentration range, 10⁹–10⁵ copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45 ± 0.31% and 1.30 ± 0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV.     

Multiplex PCR and multiplex RT-PCR for inclusive detection of major swine DNA and RNA viruses in pigs with multiple infections

Multiplex PCR and multiplex RT-PCR were developed to identify nine viruses in pigs with multiple infections. These viruses are: porcine circovirus type 2 (PCV2), suid herpesvirus 1, porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus, porcine rotavirus A (PoRV-A), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and Getah virus. These methods were shown to be high specificity and sensitivity. In the clinical application, a total of 75 field samples were examined by our methods and previously reported methods for PCV2, PRRSV, TGEV, and PEDV. As a result, the detection rates of our multiplex PCR and multiplex RT-PCR were higher than those of the previously reported methods. Furthermore, it was confirmed that 24 PCV2 positive samples were co-infected with other viruses, 11 with PRRSV, 10 with PPV, 2 with PoRV-A, and 1 with TGEV by a combination of multiplex PCR and multiplex RT-PCR. PPV and PoRV-A were newly detected by multiplex PCR and multiplex RT-PCR. These results suggest that the combination of our multiplex PCR and multiplex RT-PCR is useful for rapid and accurate identification of nine major pathogenic viruses in pigs with multiple infections.     

Clinical evaluation of a single-reaction real-time RT-PCR for pan-dengue and chikungunya virus detection

BackgroundDengue virus (DENV) and chikungunya virus (CHIKV) now co-circulate throughout tropical regions of the world, with billions of people living at risk of infection. The differentiation of these infections is important for epidemiologic surveillance as well as clinical care, though widely-used molecular diagnostics for DENV and CHIKV require the performance of two to four separate PCR reactions for detection.ObjectivesIn the current study, we sought to develop and evaluate a single-reaction, multiplex real-time RT-PCR (rRT-PCR) for the detection and differentiation of DENV and CHIKV (the pan-DENV–CHIKV rRT-PCR).Study designFrom an alignment of all available CHIKV complete genome sequences in GenBank, a new CHIKV rRT-PCR was designed for use in multiplex with a previously described assay for pan-DENV detection. Analytical evaluation was performed in accordance with published recommendations, and the pan-DENV–CHIKV rRT-PCR was clinically compared to reference molecular diagnostics for DENV and CHIKV using 182 serum samples from suspected cases in Managua, Nicaragua.ResultsThe pan-DENV–CHIKV rRT-PCR had a dynamic range extending from 7.0 to 2.0 log₁₀ copies/μL for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9–37.4 copies/μL. The pan-DENV–CHIKV rRT-PCR detected DENV in 81 patients compared to 75 using a reference, hemi-nested DENV RT-PCR, and it demonstrated perfect agreement with a reference CHIKV rRT-PCR (54 positive samples).ConclusionsThe single-reaction, multiplex format of the pan-DENV–CHIKV rRT-PCR, combined with sensitive detection of both viruses, has the potential to improve detection while decreasing testing costs and streamlining molecular workflow.     

三种呼吸道病毒实验室检测方法比较

目的在3种呼吸道多病原检测方法中筛选出敏感的实验室检测方法。方法以73份上呼道感染病例咽拭子标本作为检测对象,利用3种呼吸道多病原病毒检测方法,对17种呼吸道病毒检测指标进行平行检测,根据不同呼吸道病毒检测指标检出率,评价3种试剂检测效果。结果自建RT-PCR及PCR检测方法共检出56个阳性指标,市售多重RT-PCR检测试剂盒共检出41个阳性指标,市售多重实时荧光RT-PCR检测试剂盒共检出87个阳性指标。结论多重实时荧光RT-PCR检测试剂盒具有更高的检出率,可用于呼吸道多病原病毒的实验室检测。     

Multiplex real-time RT-PCR detection of three viruses associated with the bovine respiratory disease complex

Bovine respiratory disease complex (BRDC) is considered the most significant illness associated with feedlot cattle in North America and possibly worldwide. BRDC is a multi-factorial disease with environmental conditions interacting with multiple viral and bacterial pathogens to produce severe respiratory illness. Bovine herpesvirus 1, bovine viral diarrhoea virus and bovine parainfluenza virus 3 are three of the major viruses associated with BRDC. In this study, a multiplex real-time RT-PCR using Taqman primers and probes was developed to detect simultaneously all three of these important BRDC viruses. The assay was optimised and validated using cell-culture infected material and bovine clinical samples from BRDC cases. The sensitivity of the assay was analysed by comparing the multiplex with the individual singleplex assays, which demonstrated that viral detection was not inhibited, by multiplexing the real-time RT-PCRs. This technique is the first reported real-time PCR method for BPIV-3 and the first multiplex designed to detect three BRDC viral pathogens.     

4株蚊媒病毒多重RT-PCR快速检测方法的建立

目的建立登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒的多重RT-PCR快速检测方法。方法参照病毒核酸序列设计多重RT-PCR引物,并检索GenBank国际基因序列数据库初步验证其特异性,随后对Mg2＋、dNTP及引物浓度,RT-PCR反应条件进行优化,建立稳定、特异的多重RT-PCR快速检测4株病毒方法 ,并以同属于黄病毒科的登革3型病毒、登革4型病毒及甲病毒属基孔肯亚病毒、辛德毕斯病毒为对照,验证其特异性。结果应用多重RT-PCR反应体系,对引物的相关性实验结果表明引物之间不会因相互干扰而出现假阳性结果 ;采用多重RT-PCR引物对登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒进行扩增,分别获得574、251、879、422 bp片段,与设计相符,而对照病毒组均无非特异性扩增条带。结论实验证明,所建立的多重RT-PCR方法能够快速地检测登革1、2型病毒、黄热病毒及流行性乙型脑炎病毒,为其检测提供了一种方便易行的方法 。     

Simultaneous detection of respiratory viruses in children with acute respiratory infection using two different multiplex reverse transcription-PCR assays

A 4-tube multiplex RT-PCR (mRT-PCR), which showed higher sensitivity over conventional methods, was previously developed for the diagnosis of 14 viral pathogens of the respiratory tract. Herein the mRT-PCR was compared to the commercial Luminex mPCR-microsphere flow cytometry assay (Resplex II) which allows the detection of 12 different viruses. Eleven different viruses were identified in 91 nasopharyngeal swabs of children with acute respiratory infection, influenza A (IAV) and B, respiratory syncytial virus (RSV), human rhinovirus (hRhV), human echovirus, parainfluenza viruses (PIV) 1, 2, 3 and 4, human metapneumovirus (hMPV), and human coronavirus NL63. The results of the two techniques showed 53 and 40 positive patients by the Resplex II assay and mRT-PCR, respectively, with a concordance in 35 positive and 33 negative patients (74.7%). Individual RT-PCR tests were performed to control viruses not simultaneously detected by the two multiplex assays. The major virus misdiagnosed by mRT-PCR was IAV whereas the major viruses misdiagnosed by Resplex II were PIV1, 3 and 4. The mRT-PCR remains a simple, rapid, and specific assay for the specific detection of respiratory viruses, and can be easily implemented with standards in clinical laboratories at a low cost.     

Immune responses of poultry to Newcastle disease virus

Newcastle disease (ND) remains a constant threat to poultry producers worldwide, in spite of the availability and global employment of ND vaccinations since the 1950s. Strains of Newcastle disease virus (NDV) belong to the order Mononegavirales, family Paramyxoviridae, and genus Avulavirus, are contained in one serotype and are also known as avian paramyxovirus serotype-1 (APMV-1). They are pleomorphic in shape and are single-stranded, non-segmented, negative sense RNA viruses. The virus has been reported to infect most orders of birds and thus has a wide host range. Isolates are characterized by virulence in chickens and the presence of basic amino acids at the fusion protein cleavage site. Low virulent NDV typically produce subclinical disease with some morbidity, whereas virulent isolates can result in rapid, high mortality of birds. Virulent NDV are listed pathogens that require immediate notification to the Office of International Epizootics and outbreaks typically result in trade embargos. Protection against NDV is through the use of vaccines generated with low virulent NDV strains. Immunity is derived from neutralizing antibodies formed against the viral hemagglutinin and fusion glycoproteins, which are responsible for attachment and spread of the virus. However, new techniques and technologies have also allowed for more in depth analysis of the innate and cell-mediated immunity of poultry to NDV. Gene profiling experiments have led to the discovery of novel host genes modulated immediately after infection. Differences in virus virulence alter host gene response patterns have been demonstrated. Furthermore, the timing and contributions of cell-mediated immune responses appear to decrease disease and transmission potential. In view of recent reports of vaccine failure from many countries on the ability of classical NDV vaccines to stop spread of disease, renewed interest in a more complete understanding of the global immune response of poultry to NDV will be critical to developing new control strategies and intervention programs for the future.     

HLA class I and class II genotyping in patients with Behcet's disease: a regional study of eastern part of Turkey

Class I human leucocyte antigen (HLA)-B51 is well known to be associated with Behcet's disease in many ethnic groups. However, there has been no published paper with respect to its association with HLA class I and class II among the Turkish people who live in the eastern region of Turkey. Moreover, as it is known that B51 antigen is encoded by 21 alleles, B*5101-5121, HLA-B51 allele typing was performed, as well as HLA class I and class II genotyping of 75 patients with the disease and the 54 individuals in the matched control group. The result shows that the HLA-B51 frequency was significantly higher (58.66%) in the patient group, compared to that in the control group (18.51%) (OR = 6.245). In the subtyping of B51 alleles, 44 B51-positive patients possessed B*5101 (45.5%), B*5108 (25%), B*5105 (9.1%) and B*5104 (4.5%). There was no significant difference in the HLA-B51 allelic distribution between the patient group and the control group. However, homozygous carriers of HLA-B51 showed considerably high risk (OR = 2.647) in the patient group, compared to that in the control group. In the genotyping of class II HLA alleles, while HLA-DRB1*04 (45.3%) and HLA-DRB1*07 (24%) were the predominant alleles in the patient group, DRB1*11 (50%) appeared to be more common in the control group.     

Time course of Newcastle disease virus-induced apoptotic pathways

Newcastle disease virus (NDV) causes economically significant Newcastle disease (ND) in almost all birds worldwide. Previous studies have shown that NDV induces caspase dependent apoptotic pathways in infected cells. In the present study, time course induction of apoptotic pathways in Vero cells is described. In NDV-infected cells, caspase-8 activity, percentage of cells showing TRAIL expression was higher at 24 h p.i. (post-infection) compared to 48 h p.i. In contrast, caspase-9 activity, efflux of cytochrome c, loss of mitochondrial membrane potential was higher at 48 h compared to 24 h p.i. The caspase-3 activity was high both times. Based on these results, it was concluded that at 24 h p.i., NDV induces apoptosis through extrinsic apoptotic pathway while at 48 h p.i. predominantly through intrinsic apoptotic pathway.