Use of a Multiplex RT-PCR Assay for Simultaneous Detection of the North American Genotype Porcine Reproductive and Respiratory Syndrome Virus,Swine Influenza Virus and Japanese Encephalitis Virus

A multiplex reverse transcriptase-polymerase chain reaction(multiplex RT-PCR) assay was developed and subsequently evaluated for its efficacy in the detection of multiple viral infections simultaneously,in swine.Specific primers for each of the 3 RNA viruses,North American genotype porcine reproductive and respiratory syndrome virus,Japanese encephalitis virus,and swine influenza virus,were used in the testing procedure.The assay was shown to be highly sensitive because it could detect as little as 10-5 ng of each of the respective amplicons in a single sample containing a composite of all 3 viruses.The assay was also effective in detecting one or more of the same viruses in various combinations in specimens,including lymph nodes,lungs,spleens,and tonsils,collected from clinically ill pigs and in spleen specimens collected from aborted pig fetuses.The results from the multiplex RT-PCR were confirmed by virus isolation.The relative efficiency(compared to the efficiency of separate assays for each virus) and apparent sensitivity of the multiplex RT-PCR method show that this method has potential for application in routine molecular diagnostic procedures.

Simultaneous Detection of Three Arboviruses Using a Triplex RT-PCR Enzyme Hybridization Assay

Arboviruses represent a serious problem to public health and agriculture worldwide. Fast, accurate identification of the viral agents of arbovirus-associated disease is essential for epidemiological surveillance and laboratory investigation. We developed a cost-effective, rapid, and highly sensitive one-step "triplex RT-PCR enzyme hybridization" assay for simultaneous detections of Japanese Encephallitis virus (JEV, Flaviviridae), Getah virus (GETV, Togaviridae), and Tahyna virus (TAHV, Bunyaviridae) using three pairs of primers to amplify three target sequences in one RT-PCR reaction. The analytical sensitivity of this assay was 1 PFU/mL for JEV, 10 PFU/mL for GETV, and 10 PFU/mL for TAHV. This assay is significantly more rapid and less expensive than the traditional serological detection and single RT-PCR reaction methods. When "triplex RT-PCR enzyme hybridization" was applied to 29 cerebrospinal fluid (CSF) samples that were JEV-positive by normal RT-PCR assay, all samples were strongly positive for JEV, but negative for GETV and TAHV, demonstrating a good sensitivity, specificity, and performance at CSF specimen detection.

Multiplex real-time RT-PCR for detecting chikungunya virus and dengue virus

Objective:To develop diagnostic test for detection chikungunya virus(CHIKV and Dengue virus (DENV) infection.Methods:We have performed a rapid,accurate laboratory confirmative method to simultaneously detect,quantify and differentiate CHIKV and DENV infection by single-step multiplex real-time RT-PCR.Results:The assay’s sensitivity was 97.65%,specificity was 92.59% and accuracy was 95.82%when compared to conventional RT-PCR.Additionally,there was no cross-reaction between CHIKV,DENV,Japanese encephalitis virus,hepatitis C,hepatitis A or hepatitis E virus.Conclusions:This rapid and reliable assay provides a means for simultaneous early diagnosis of CHIKV and DENV in a single-step reaction.

Biological and molecular characterization of tomato brown rugose fruit virus and development of quadruplex RT-PCR detection

Tomato brown rugose fruit virus(ToBRFV) is a novel tobamovirus firstly reported in 2015 and poses a severe threat to the tomato industry. So far, it has spread to 10 countries in America, Asia, and Europe. In 2019, ToBRFV was identified in Shandong Province(ToBRFV-SD), China. In this study, it was shown that ToBRFV-SD induced mild to severe mosaic and blistering on leaves, necrosis on sepals and pedicles, and deformation, yellow spots, and brown rugose necrotic lesions on fruits. ToBRFV-SD induced distinct symptoms on plants of tomato, Capsicum annumm, and Nicotiana benthamiana, and caused latent infection on plants of Solanum tuberosum, Solanum melongena, and N. tabacum cv. Zhongyan 102. All the 50 tomato cultivars tested were highly sensitive to ToBRFV-SD. The complete genomic sequence of ToBRFV-SD shared the highest nucleotide and amino acid identities with isolate IL from Israel. In the phylogenetic tree constructed with the complete genomic sequence, all the ToBRFV isolates were clustered together and formed a sister branch with tobacco mosaic virus(TMV). Furthermore, a quadruplex RT-PCR system was developed that could differentiate ToBRFV from other economically important viruses affecting tomatoes, such as TMV, tomato mosaic virus, and tomato spotted wilt virus. The findings of this study enhance our understanding of the biological and molecular characteristics of ToBRFV and provide an efficient and effective detection method for multiple infections, which is helpful in the management of ToBRFV.

Real-time RT-PCR Assay for the detection of Tahyna Virus

A real-time RT-PCR （RT-qPCR） assay for the detection of Tahyna virus was developed to monitor Tahyna virus infection in field-collected vector mosquito samples. The targets selected for the assay were S segment sequences encoding the nucleocapsid protein from the Tahyna virus. Primers and probes were selected in conserved regions by aligning genetic sequences from various Tahyna virus strains available from GenBank. The sensitivity of the RT-qPCR approach was compared to that of a standard plaque assay in BHK cells. RT-qPCR assay can detect 4.8 PFU of titrated Tahyna virus. Assay specificities were determined by testing a battery of arboviruses, including representative strains of Tahyna virus and other arthropod-borne viruses from China. Seven strains of Tahyna virus were confirmed as positive; the other seven species of arboviruses could not be detected by RT-qPCR. Additionally, the assay was used to detect Tahyna viral RNA in pooled mosquito samples. The RT-qPCR assay detected Tahyna virus in a sensitive, specific, and rapid manner; these findings support the use of the assay in viral surveillance.

Tomato mottle mosaic virus: Characterization, resistance gene effectiveness, and quintuplex RT-PCR detection system

Tomato mottle mosaic virus(ToMMV), an economically important species of the genus Tobamovirus, causes significant loss in yield and quality of tomato fruits. Here, we identified the Shandong isolate of ToMMV(ToMMV-SD) collected from symptomatic tomato fruits in Weifang, Shandong Province of China. ToMMV-SD caused symptoms such as severe mosaic, mottling, and necrosis of tomato leaves, yellow spot and necrotic lesions on tomato fruits. The obtained full genome of ToMMV-SD was 6 399 nucleotides(accession number MW373515) and had the highest identity of 99.5% with that of isolate SC13-051 from the United States of America at the genomic level. The infectious clone of ToMMV-SD was constructed and induced clear mosaic and necrotic symptoms onto Nicotiana benthamiana leaves. Several commercial tomato cultivars, harboring Tm-2~2 resistance gene, and pepper cultivars, containing L resistance gene, were susceptible to ToMMV-SD. Plants of Solanum melongena(eggplant) and Brassica pekinensis(napa cabbage) showed mottling symptoms, while N. tabacum cv. Zhongyan 100 displayed latent infection. ToMMV-SD did not infect plants of N. tabacum cv. Xanthi NN, Brassica rapa ssp. chinensis(bok choy), Raphanus sativus(radish), Vigna unguiculata cv. Yuanzhong 28-2(cowpea), or Tm-2~2 transgenic N. benthamiana. A quintuplex RT-PCR system differentiated ToMMV from tomato mosaic virus, tomato brown rugose fruit virus, tobacco mosaic virus, and tomato spotted wilt virus, with the threshold amount of 0.02 pg. These results highlight the threat posed by ToMMV to tomato and pepper cultivation and offer an efficient detection system for the simultaneous detection of four tobamoviruses and tomato spotted wilt virus infecting tomato plants in the field.

Designing Primers for H5 and H7 Subtypes of Avian Influenza Virus and Multiplex RT-PCR Amplification

[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.

Subtyping Animal Influenza Virus with General Multiplex RT-PCR and Liquichip High Throughput (GMPLex)

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of ill, H3, H5, HT, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR （GM RT-PCR）. It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus （IV） rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features： high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10-5（= 280ELDs0） forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10-4（=2800 ELD50）. The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.

Development of a multiplex one-step real-time RT-PCR assay for the simultaneous detection of eight viruses associated with febrile rash illnesses

Fever and rash illnesses(FRIs)are a series of common diseaseswith fever and rashes as clinicalmanifestations,most of which are caused by viral infection.The rashes of FRIs are generally nonspecific;therefore it is difficult to identify FRIassociated viruses solely based on clinical symptoms.To achieve rapid and accurate identification of FRI pathogens,a multiplex one-step real-time reverse transcription-polymerase chain reaction(RT-PCR)assay was developed and evaluated in this study.Primers and probes were selected for the detection of measles virus(MeV),rubella virus(RV),human enterovirus(EV),varicella-zoster virus(VZV),dengue virus(DENV),human parvovirus B19(B19),Epstein-Barr virus(EBV),and human herpes virus 6(HHV-6),which cover the most common pathogenic viruses of FRIs.Detection of the eight FRI-associated viruses,which was divided into two groups/tubes,was simultaneously performed under universal optimized reaction conditions in multiplex one-step real-time RT-PCR assay.The multiplex realtime RT-PCR showed high sensitivity and specificity in detecting the eight FRI-associated viruses.The limits of detection(LODs)for the eight viruses were in the range of 47–177 copies/reaction,and no cross reactions for the eight FRIassociated viruses were found in the multiplex assay.In addition,the results of the multiplex real-time RT-PCR assay were consistent with the results of a monoplex real-time RT-PCR assay and sequencing for clinical specimens obtained from FRI patients.With its advantages of high efficiency and rapid and accurate diagnosis,multiplex real-time RT-PCR was very feasible for the early diagnosis of FRI pathogenic viruses and would be of great help for the proper treatment,monitoring,and initiation of preventive measures for FRI cases.

应用多重RT-PCR检测甘蔗黄叶病毒和高粱花叶病毒

建立了多重RT-PCR同时检测甘蔗黄叶病毒(Sugarcane yellow leaf virus,ScYLV)和高粱花叶病毒病毒(Sorghummosaic virus,SrMV)的技术体系,该体系可有效地对甘蔗田间植株和试管苗进行检测。根据引物之间的互补性及引物的Tm值筛选多重PCR引物。确定适宜的PCR缓冲液的浓度为1×,退火温度为53℃,延伸温度为72℃。

侵染甘薯的SPCSV、SPVG、SPFMV多重RT-PCR检测方法的建立及应用

为建立可同时检测甘薯褪绿矮化病毒（SPCSV）、甘薯G病毒（SPVG）和甘薯羽状斑驳病毒（SPFMV）多重RT-PCR检测方法,本文根据SPCSV热激蛋白基因（hsp70）及SPVG、SPFMV外壳蛋白基因（CP）基因核苷酸序列的保守区域设计特异性引物,通过引物筛选,优化多重RT-PCR反应条件,建立了能同时检测SPCSV、SPVG和SPFMV 3种病毒的多重RT-PCR检测方法。该体系能有效扩增出大小为304、433、601 bp 3个特异性片段。测序结果表明3种病毒与参考序列的一致性达94%-99%。应用建立的多重RT-PCR检测方法可稳定、准确、灵敏地同时检测单一或复合侵染3种甘薯病毒,为甘薯脱毒和病毒病诊断奠定基础。

三重RT-PCR同步检测马铃薯多种病毒影响因素

根据病毒外壳蛋白区序列设计PVX、PVS特异性引物对 ,根据P1基因区序列设计PVA特异性引物对 ,应用三重RT PCR同步检测马铃薯X病毒 ,马铃薯A病毒及马铃薯S病毒 ,分别得到 5 62bp、 2 5 5bp、 1 82bp大小的扩增片段。试验从反转录反应、PCR反应及循环条件 3方面讨论了试剂和循环条件对三重RT PCR同步检测 3种病毒的影响。结果表明反转录反应中dNTPs浓度、 3种病毒下游引物浓度比例对整个反应影响较大 ;其次是PCR反应中MgCl2 浓度和退火温度 ;反转录时间 ,循环条件对RT PCR影响较小。

利用多重RT-PCR技术检测草莓病毒的研究

Coat protein gene of 12 isolates of Strawberry vein banding virus(SVBV) was studied by multiple sequence alignment and the primers located in conserved region were designed.The detection protocol for SVBV by reverse transcriptase polymerase chain reaction(RT-PCR) was developed.The primers of multiplex RT-PCR were selected by primer-primer interactions and the melting temperature.The annealing temperature,the concentration of PCR buffer,the extension temperature,the extension time and the concentration of pri-mers were optimized,respectively.A multiplex RT-PCR assay was made for simultaneous detecting Strawberry mottle virus(SMoV),Strawberry mild yellow edge virus(SMYEV) and SVBV.Both field-grown strawberries and microplants were detected effectively.It was the first report that multiplex RT-PCR was used to assay the efficacy of strawberry viruses elimination.

Detection of Citrus yellow vein clearing virus by Quantitative Real-time RT-PCR

To develop a rapid and reliable detection method for Citrus yellow vein clearing virus(CYVCV), a quantitative real-time reverse transcriptionpolymerase chain reaction(q RT-PCR) system based on SYBR Green I was established by using a pair of specific primers designed from its conserved coat protein gene. The sensitivity, specificity, and applicability of the system were evaluated accordingly. The results showed that amplicons were produced from CYVCV isolates, whereas no amplicons from non-CYVCV citrus virus samples, including Citrus tristeza virus(CTV) and Citrus tatter leaf virus(CTLV), were obtained. The sensitivity of the q RT-PCR was 100-fold higher than that of conventional RT-PCR. An excellent linear correlation(R2= 0.999) was obtained from two standard curves of c RNA, and the amplification efficiency was 102%. The data from field citrus samples detection showed that the q RT-PCR system could be used in determining the concentration of CYVCV in different citrus species.

Detection of Three Virus Diseases and Their Distribution in Xinjiang Melon Region using Multiplex RTPCR Technique

Using homologous cloning method, partial fragments of coat protein （CP） gene of WMV, CMV and ZYMV were cloned from virus-infected melon in Xinjiang. The reaction system of multiplex RT-PCR was optimized based on singleplex RT-PCR amplification conditions, using single factor analysis. Forth-eight samples were tested separately with multiplex RT-PCR. The results showed that both assays run to consistent results. The optimized multiplex RT-PCR system had certain accuracy and stability, and could be used for quick detection, pathogen identification and positive screening of WMV （ Watermelon mosaic virus）, CMV （ Cucumber mosaic virus）, and ZYMV （Zucchini yellow mosaic virus）. The distribution status and infection form of three kinds of viruses was determined in main melon growing area of Xinjiang, providing theoretical foundation and experimental evidence for virus diseases control, field testing, epidemiological investigation and melon virus-resistant breeding in Xinjiang.

A quantitative RT-PCR assay for rapid detection of Eurasianlineage H10 subtype influenza A virus

Dear Editor,Influenza A viruses(IAVs)are single-stranded,negative sense RNA viruses.IAV subtype is determined on the basis of the viral surface glycoproteins,hemagglutinin(HA),and neuraminidase(NA).To date,18 HA and 11NA subtypes have been reported(Tong et al.,2012).

4种茉莉病毒一步法多重RT-PCR检测技术的建立

茉莉生长过程中普遍受到多种病毒的复合侵染。由于缺乏病毒与症状关系的系统研究,仅通过症状很难准确分辨茉莉被何种病毒侵染,所以建立快速精准诊断多种茉莉病毒病的检测技术具有重要应用价值。本研究建立了一种可同时检测4种茉莉病毒——茉莉T病毒(jasmine virus T,JaVT)、茉莉C病毒(jasmine virus C,JaVC)、茉莉H病毒(jasminevirus H,JaVH)和茉莉A病毒(jasmine virus A,JaVA)——的一步法多重逆转录PCR(one-step multiplex reverse transcription-PCR,RT-PCR)体系,其最优反应体系为:在总体系20.0μL中,JaVT、JaVC、JaVH和JaVA的上下游引物浓度分别为200、150、100和150nmol·L^(-1),One-step Enzyme Mix:0.2μL,2x反应缓冲液:10.0μL,模板量:1.0μg;多重RT-PCR参数设定为:RNA反转录50℃30min;预变性94℃2min;94℃变性30s;55℃退火30s;72℃延伸45s,30个循环;72℃整体延伸10 min。研究结果表明建立的一步法多重RT-PCR可同时高效精准地检测4种茉莉病毒,极大地提高了检测效率,可被广泛应用于实验室精准、高效检测和田间茉莉病毒病的检测。

Development of a duplex real-time PCR method for the detection of influenza C and D viruses

Influenza viruses are major respiratory pathogens known to infect human and a variety of animals and are widely prevalent worldwide.Genome structure of influenza D virus(IDV)is identical to that of influenza C virus(ICV),and phylogenetic analyses suggest that IDV and ICV share a common ancestry and high homology.To date,the prevalence of ICV and IDV in China is unclear,but these viruses represent a potential threat to public health due to cross-species transmission and zoonotic potential.To efficiently monitor ICV and IDV,it is necessary to establish a dual detection method to understand their prevalence and conduct in-depth research.A duplex real-time PCR method for the simultaneous detection of ICV and IDV was developed.TaqMan fluorescent probes and specific primers targeting NP gene of ICV and PB1 gene of IDV were designed.This method exhibited good specificity and sensitivity,and the detection limit reached 1 × 10^(1) copies/pL of plasmid standards of each pathogen.Thirty-one clinical swine samples and 10 clinical cattle samples were analyzed using this method.One positive sample of IDV was detected,and the accuracy of clinical test results was verified by conventional PCR and DNA sequencing.The duplex real-time PCR detection method represents a sensitive and specific tool to detect IG/and IDV,It provides technical support for virus research and clinical diagnosis of ICV and IDV.This information will benefit animal and human health.

A sensitive SYBR Green RT-qPCR method for grapevine virus E and its application for virus detection in different grapevine sample types

To develop a rapid and high-sensitivity method for detection of grapevine virus E(GVE),a SYBR Green based real-time fluorescence quantitative RT-PCR method(RT-qPCR)was established.This method could be used to detect GVE specifically,and the sensitivity was about 100 times greater than conventional RT-PCR.An excellent linear correlation(R=0.997)and a high amplification efficiency(E=97.5%)were obtained from the standard curve of this method.Reproducibility tests revealed that the coefficients of variation in the intra-and inter-assay results were 0.31-1.03%and 0.82--262%,respectively,indicating a good reproduiblity.The RT-qPCR method could be used to detect GVE in a wide range of grapevine sample types.The detection rates of RT-qPCR for nearly all sample types from different positions and seasons were higher than conventional RT-PCR.The detection rates in spring,summer,autumn and winter increased gradually.Samples in autumn and winter were best for detection,and the detection rates of most samples were 80-100%,which were 10 to 40%higher than conventional RT-PCR.In general,old petioles and branches were the best tissues for GVE detection.The detection rates of these samples in each season were all 100%,which were 20 to 40%higher than conventional RT-PCR.The second highest rates were in the old leaf,with detection rates for RT-qPCR of 80-100%in all seasons,which were 20 to 40%higher than conventional RT-PCR.GVE could be difficultly detected in young leaves by conventional RT-PCR,and the detection rates were only 0-50%,while by RT-qPCR the rates could increase to 0--80%.A total of 33 out of 363 samples(belonging to 68 cultivars)from 20 regions in China were detected to be positive by RT-qPCR(9.1%),which was more than twice the rate of the conventional RT-PCR(3.9%).

A Pair of Novel Primers for Universal Detection of the NS1 Gene from Various Bluetongue Virus Serotypes

Twenty five serotypes of Bluetongue virus (BTV) have been identified worldwide. Rapid and reliable methods of virus universal detection are essential for fighting against bluetongue (BT). We have therefore developed and evaluated a pair of primers which can detect various serotypes of BTV by RT-PCR. Analysis of the viral protein 7 (VP7) and the non-structural protein (NS1) gene from different serotypes of BTV by DNAstar showed that the 5' end of the NS1 gene is the most conserved region. The primer pairs (P1 and P2) were designed based on the highly conserved region of NS1. The novel primers were evaluated by detecting BTV serotypes 1, 3, 5, 8, 10, 11, 21 and 22. The specificity of the primers was estimated by comparing to gene sequences of viruses published in GenBank, and further assessed by detecting BTV serotype 1-12 and Epizootic hemorrhagic disease virus (EHDV) serotype 1-4. The sensitivity and repeatability of PCR with the novel primers were evaluated by successfully detecting the recombinant plasmid pGEM-T121 containing the diagnosed nucleotide sequence. Our results suggest that these unique primers can be used in high throughout and universal detection of the NS1 gene from various BTV serotypes.