Development of a multiplex RT‐PCR for the detection of major diarrhoeal viruses in pig herds in China

The major enteric RNA viruses in pigs include porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus A (PRV‐A), porcine kobuvirus (PKV), porcine sapovirus (PSaV) and porcine deltacoronavirus (PDCoV). For differential diagnosis, a multiplex RT‐PCR method was established on the basis of the N genes of TGEV, PEDV and PDCoV, the VP7 gene of PRV‐A, and the polyprotein genes of PKV and PSaV. This multiplex RT‐PCR could specifically detect TGEV, PEDV, PDCoV, PRV‐A, PKV and PSaV without cross‐reaction to any other major viruses circulating in Chinese pig farms. The limit of detection of this method was as low as 10⁰–10¹ ng cDNA of each virus. A total of 398 swine faecal samples collected from nine provinces of China between October 2015 and April 2017 were analysed by this established multiplex RT‐PCR. The results demonstrated that PDCoV (144/398), PSaV (114/398), PEDV (78/398) and PRV‐A (70/398) were the main pathogens, but TGEV was not found in the pig herds in China. In addition, dual infections, for example, PDCoV + PSaV, PDCoV + PRV‐A, PRA‐V + PSaV and PEDV + PDCoV, and triple infections, for example, PDCoV + PRV‐A + PSaV and PEDV + PDCoV + PKV, were found among the collected samples. The multiplex RT‐PCR provided a valuable tool for the differential diagnosis of swine enteric viruses circulating in Chinese pig farms and will facilitate the prevention and control of swine diarrhoea in China.     

Development of universal and quadruplex real‐time RT‐PCR assays for simultaneous detection and differentiation of porcine reproductive and respiratory syndrome viruses

Porcine reproductive and respiratory syndrome virus 1 (PRRSV1) and 2 (PRRSV2) (including 3 major subtypes: classical (CA‐PRRSV2), highly pathogenic (HP‐PRRSV2) and NADC30‐like (NL‐PRRSV2)) are currently coexisting in Chinese swine herds but with distinct virulence. Reliable detection and differentiation assays are crucial to monitor the prevalence of PRRSV and to adopt effective control strategies. However, current diagnostic methods cannot simultaneously differentiate the four major groups of PRRSV in China. In this study, universal and quadruplex real‐time RT‐PCR assays using TaqMan‐MGB probes were developed for simultaneous detection and differentiation of Chinese PRRSV isolates. The newly developed real‐time RT‐PCR assays exhibited good specificity, sensitivity, repeatability and reproducibility. In addition, the newly developed real‐time RT‐PCR assays were further validated by comparing with a universal PRRSV conventional RT‐PCR assay on the detection of 664 clinical samples collected from 2016 to 2019 in China. Based on the clinical performance, the agreements between the universal and quadruplex real‐time RT‐PCR assays and the conventional RT‐PCR assay were 99.55% and 99.40%, respectively. Totally 90 samples were detected as PRRSV‐positive, including 2 samples that were determined to be co‐infected with NL‐PRRSV2 and HP‐PRRSV2 isolates by the quadruplex real‐time RT‐PCR assay. ORF5 sequencing confirmed the real‐time RT‐PCR results that 2, 6, 27 and 57 of the 92 sequences were PRRSV1, CA‐PRRSV2, NL‐PRRSV2 and HP‐PRRSV2, respectively. This study provides promising alternative tools for simultaneous detection and differentiation of PRRSV circulating in Chinese swine herds.     

Development of Real‐Time RT‐PCR Assays for Detection and Typing of Epizootic Haemorrhagic Disease Virus

Epizootic haemorrhagic disease virus (EHDV) is an emerging arboviral pathogen of wild and domestic ruminants worldwide. It is closely related to bluetongue virus (BTV) and is transmitted by adult females of competent Culicoides vector species. The EHDV genome consists of ten linear double‐stranded (ds)RNA segments, encoding five non‐structural and seven structural proteins. Genome‐segment reassortment contributes to a high level of genetic variation in individual virus strains, particularly in the areas where multiple and distinct virus lineages co‐circulate. In spite of the relatively close relationship between BTV and EHDV herd‐immunity to BTV does not appear to protect against the introduction and infection of animals by EHDV. Although EHDV can cause up to 80% morbidity in affected animals, vaccination with the homologous EHDV serotype is protective. Outer‐capsid protein VP2, encoded by Seg‐2, is the most variable of the EHDV proteins and determines both the specificity of reactions with neutralizing antibodies and consequently the identity of the eight EHDV serotypes. In contrast, VP6 (the viral helicase), encoded by Seg‐9, is highly conserved, representing a virus species/serogroup‐specific antigen. We report the development and evaluation of quantitative (q)RT‐PCR assays targeting EHDV Seg‐9 that can detect all EHDV strains (regardless of geographic origin/topotype/serotype), as well as type‐specific assays targeting Seg‐2 of the eight EHDV serotypes. The assays were evaluated using orbivirus isolates from the ‘Orbivirus reference collection’ (ORC) at The Pirbright Institute and were shown to be EHDV pan‐reactive or type‐specific. They can be used for rapid, sensitive and reliable detection and identification (typing) of EHDV RNA from infected blood, tissue samples, homogenized Culicoides, or tissue culture supernatant. None of the assays detected RNA from closely related but heterologous orbiviruses, or from uninfected host animals or cell cultures. The techniques presented could be used for both surveillance and vaccine matching (serotype identification) as part of control strategies for incursions in wild and domestic animal species.     

Detection of Micrometastases in Peritoneal Washings of Pancreatic Cancer Patients by the Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVE: Pancreatic cancer patients with positive (+) peritoneal cytology have a prognosis similar to stage IV patients. We studied the ability of quantitative real time-polymerase chain reaction (RT-PCR) to detect micrometastases in patients undergoing staging laparoscopy. METHODS: Peritoneal washes were obtained prospectively from 35 consecutive patients with pancreatic adenocarcinoma undergoing staging laparoscopy and 16 patients undergoing laparoscopy for benign disease. Each sample was assessed by cytologic examination and RT-PCR analysis for tumor markers: CEA, CK7, Kras2, and MUC1. Markers and their combinations were evaluated on the basis of their deviance from the ideal marker. RESULTS: Pathologic stages for pancreatic cancer patients were: 1A-1 (3%), IB-1 (3%), IIA-5 (15%), IIB-13 (38%), III-5 (15%), IV-9 (26%). Eight patients were cytology (+) and stages IIA-1, IIB-2, IV-5. Twenty-five patients were RT-PCR (+). The optimal threshold for cycle amplification was 35 based on a receiver operating characteristic curve. CEA had the best profile of sensitivity, specificity, PPV, NPV, and the smallest deviance. CONCLUSION: RT-PCR using a panel of tumor markers, including CEA, was comparable in sensitivity, specificity, PPV, and NPV to cytology. RT-PCR could represent a more sensitive method for detection of subclinical peritoneal tumor dissemination; this may be useful in patient selection for operative management and clinical trials.     

Evidence of Presence of Mycobacterium tuberculosis in Bovine Tissue Samples by Multiplex PCR: Possible Relevance to Reverse Zoonosis

Bovine tuberculosis, caused by Mycobacterium bovis, remains one of the most important zoonotic health concerns worldwide. The transmission of Mycobacterium tuberculosis from humans to animals also occurs especially in countries where there is close interaction of humans with the animals. In the present study, thirty bovine lung tissue autopsy samples from an organized dairy farm located in North India were screened for the presence of Mycobacterium tuberculosis complex by smear microscopy, histopathological findings and PCR. Differential diagnosis of M. tuberculosis and M. bovis was made based on the deletion of mce‐3 operon in M. bovis. The present study found eight of these samples positive for M. tuberculosis by multiplex PCR. Sequencing was performed on two PCR‐positive representative samples and on annotation, and BLAST analysis confirmed the presence of gene fragment specific to Mycobacterium tuberculosis. The presence of M. tuberculosis in all the positive samples raises the possibility of human‐to‐cattle transmission and possible adaptation of this organism in bovine tissues. This study accentuates the importance of screening and differential diagnosis of Mycobacterium tuberculosis complex in humans and livestock for adopting effective TB control and eradication programmes.     

DD3 mRNA在前列腺癌组织中定量表达分析

目的:探讨DD3基因在前列腺组织中表达的临床意义。方法:用荧光定量RT-PCR方法对21例前列腺癌(PCa)组织、27例非前列腺部位的肿瘤组织、39例良性前列腺增生(BPH)组织和15例正常前列腺组织中的DD3的表达进行了定量分析,用ROC曲线对DD3 mRNA诊断PCa的性能进行了分析。结果:27例非前列腺组织中均未检测到DD3基因的表达。DD3在PCa组、BPH组和正常前列腺组表达量的中位数分别为7.2×106、2.5×104、1.5×104拷贝/mg组织。PCa组较BPH组和正常前列腺组DD3的表达量显著增高(P<0.01),而BPH组和正常前列腺组间则差异无显著性(P>0.05)。DD3表达量与临床分期和分化程度之间均无明显相关性。DD3 mRNA曲线下面积(AUC-ROC)为0.937(95%CI:0.879～0.995)。当临界值为1.4×105拷贝/mg组织时,灵敏度、特异度、准确度、阳性预测值(PPV)、阴性预测值(NPV)、阳性拟然比(+LR)、阴性拟然比(-LR)分别为90.5%、85.0%、86.7%、76.0%、94.3%、6.03和0.11。结论:DD3 mRNA的表达仅限于前列腺组织,具有良好的组织特异性。DD3 mRNA表达在PCa组织中显著升高,在正常前列腺和BPH组织中差异无显著性。DD3 mRNA可作为PCa诊断的良好标志物,在PCa早期诊断、微转移诊断、预后判断、指导治疗等方面也具有潜在的应用价值。     

Rapid and simple detection of foot‐and‐mouth disease virus: Evaluation of a cartridge‐based molecular detection system for use in basic laboratories

Highly contagious transboundary animal diseases such as foot‐and‐mouth disease (FMD ) are major threats to the productivity of farm animals. To limit the impact of outbreaks and to take efficient steps towards a timely control and eradication of the disease, rapid and reliable diagnostic systems are of utmost importance. Confirmatory diagnostic assays are typically performed by experienced operators in specialized laboratories, and access to this capability is often limited in the developing countries with the highest disease burden. Advances in molecular technologies allow implementation of modern and reliable techniques for quick and simple pathogen detection either in basic laboratories or even at the pen‐side. Here, we report on a study to evaluate a fully automated cartridge‐based real‐time RT ‐PCR diagnostic system (Enigma MiniLab^®) for the detection of FMD virus (FMDV ). The modular system integrates both nucleic acid extraction and downstream real‐time RT ‐PCR (rRT ‐PCR ). The analytical sensitivity of this assay was determined using serially diluted culture grown FMDV , and the performance of the assay was evaluated using a selected range of FMDV positive and negative clinical samples of bovine, porcine and ovine origin. The robustness of the assay was evaluated in an international inter‐laboratory proficiency test and by deployment into an African laboratory. It was demonstrated that the system is easy to use and can detect FMDV with high sensitivity and specificity, roughly on par with standard laboratory methods. This cartridge‐based automated real‐time RT ‐PCR system for the detection of FMDV represents a reliable and easy to use diagnostic tool for the early and rapid disease detection of acutely infected animals even in remote areas. This type of system could be easily deployed for routine surveillance within endemic regions such as Africa or could alternatively be used in the developed world.     

Assessment of Preparation of Samples Under the Field Conditions and a Portable Real‐Time RT‐PCR Assay for the Rapid On‐Site Detection of Newcastle Disease Virus

Newcastle disease virus (NDV), also known as virulent forms of avian paramyxovirus serotype 1 (AMPV‐1), is the causative agent of Newcastle disease affecting many species of birds and causing heavy losses to the poultry industry worldwide. Early, rapid and sensitive detection of the viruses or the viral nucleic acids is very important for disease diagnosis and control. This study aimed to evaluate sample preparation under field conditions and the application of a real‐time RT‐PCR method in the portable T‐COR4 platform for the rapid, on‐site detection of NDV on a farm. In the laboratory setting, the portable real‐time RT‐PCR assay had a similar performance compared with that obtained with a larger, stationary Rotor Gene real‐time thermocycler. In the field conditions, viral nucleic acids were manually extracted just outside of animal units with minimal equipment and real‐time RT‐PCR detection was performed with the portable thermocycler T‐COR4 placed in a nearby room. The portable assay at the farm detected viral RNA in 15 samples and reached an agreement of 83% (39/47) when the same RNA preparations were tested in the Rotor Gene thermocycler under the laboratory setting. The results demonstrated the feasibility of performing field detection but also the need to improve and further simplify sample preparation procedures.     

Post‐epidemic investigation of Schmallenberg virus in wild ruminants in Slovenia

Schmallenberg virus (SBV) is a vector‐borne virus belonging to the genus Orthobunyavirus within the Bunyaviridae family. SBV emerged in Europe in 2011 and was characterized by epidemics of abortions, stillbirths and congenital malformations in domestic ruminants. The first evidence of SBV infection in Slovenia was from an ELISA‐positive sample from a cow collected in August 2012; clinical manifestations of SBV disease in sheep and cattle were observed in 2013, with SBV RNA detected in samples collected from a total of 28 herds. A potential re‐emergence of SBV in Europe is predicted to occur when population‐level immunity declines. SBV is also capable of infecting several wild ruminant species, although clinical disease has not yet been described in these species. Data on SBV‐positive wild ruminants suggest that these species might be possible sources for the re‐emergence of SBV. The aim of this study was to investigate whether SBV was circulating among wild ruminants in Slovenia and whether these species can act as a virus reservoir. A total of 281 blood and spleen samples from wild ruminants, including roe deer, red deer, chamois and European mouflon, were collected during the 2017–2018 hunting season. Serum samples were tested for antibodies against SBV by ELISA; the overall seroprevalence was 18.1%. Seropositive samples were reported from all over the country in examined animal species from 1 to 15 years of age. Spleen samples from the seropositive animals and serum samples from the seronegative animals were tested for the presence of SBV RNA using real‐time RT‐PCR; all the samples tested negative. Based on the results of the seropositive animals, it was demonstrated that SBV was circulating in wild ruminant populations in Slovenia even after the epidemic, as almost half (23/51) of the seropositive animals were 1 or 2 years old.     

Opportunities for enhanced surveillance of foot‐and‐mouth disease in endemic settings using milk samples

Under‐reporting of foot‐and‐mouth disease (FMD) masks the true prevalence in parts of the world where the disease is endemic. Laboratory testing for the detection of FMD virus (FMDV) is usually reliant upon the collection of vesicular epithelium and fluid samples that can only be collected from acutely infected animals, and therefore animals with sub‐clinical infection may not be identified. Milk is a non‐invasive sample type routinely collected from dairy farms that has been utilized for surveillance of a number of other diseases. The aim of this study was to examine the application of milk as an alternative sample type for FMDV detection and typing, and to evaluate milk as a novel approach for targeted surveillance of FMD in East Africa. FMDV RNA was detected in 73/190 (38%) individual milk samples collected from naturally infected cattle in northern Tanzania. Furthermore, typing information by lineage‐specific rRT‐PCR assays was obtained for 58% of positive samples, and corresponded with the virus types identified during outbreak investigations in the study area. The VP1‐coding sequence data obtained from milk samples corresponded with the sequence data generated from paired epithelial samples collected from the same animal. This study demonstrates that milk represents a potentially valuable sample type for FMDV surveillance and might be used to overcome some of the existing biases of traditional surveillance methods. However, it is recommended that care is taken during sample collection and testing to minimize the likelihood of cross‐contamination. Such approaches could strengthen FMDV surveillance capabilities in East Africa, both at the individual animal and herd level.