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.

Respiratory Virus Multiplex RT-PCR Assay Sensitivities and Influence Factors in Hospitalized Children with Lower Respiratory Tract Infections

Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex lI V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab （NPS） specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children＇s Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system （Qiagen, Germany） and 17 respiratory viruses and genotypes including influenza A virus （FluA）, FluB, parainfluenza virus 1 （PIV1）, PIV2, PIV3, PIV4, respiratory syncytial virus （RSV）, human metapneumovirus （hMPV）, rhinoviruses （RhV）, enteroviruses （EnV）, human bocaviruses （hBoV）, adenoviruses （AdV）, four coronaviruses （229E, OC43, NL63 and HKU1）, and FluA 2009 pandemic H1NI（H1NI-p） were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 （62.6%） and 201（45.9%） specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9-100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities （11.1%-40.0%） were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H 1N 1-p and RSV （p=0.011-0.000） The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous detection of 17 viral pathogens in NPS specimens in pediatric inpatients at the time of admission. The sensitivity of multiplex RT-PCR was influenced by viral loads, specimen process methods, primer and probe design and amplification condition.

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.

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.

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.

Rapid Detection Co-infections of Classical Swine Fever Virus and Porcine Reproductive and Respiratory Syndrome Virus by One-step Multiplex RT-PCR

Classical swine fever virus （CSFV） and porcine reproductive and respiratory syndrome virus （PRRSV） have caused immense economic loss in the pig industry and are considered to be the two most important infectious diseases of pigs in the world A multiplex reverse transcription polymerase chain reaction （multiplex RT-PCR） was developed for CSFV and PRRSV co-infections or infections, respectively. A set of two pairs of primer was designed based on the sequence of nonstructural protein NS54B of CSFV and ORF7 gene of PRRSV. The diagnostic accuracy of multiplex RT-PCR assay was evaluated by using 56 field clinical samples by multiplex RT-PCR, single RT-PCR and sequence analysis; and the specificity of multiplex PCR was verified by using constructed plasmids containing the specific viral target fragments of PRRSV and CSFV, respectively. The results indicated that this assay could reliably differentiate PRRSV and CSFV in co-infection samples. The multiplex RT-PCR developed in this study might provide a new avenue to the rapid the detection of CSFV and PRRSV in one reaction.

Detection of the Mex Efflux Pumps in <i>Pseudomonas</i><i>aeruginosa</i>by Using a Combined Resistance-Phenotypic Markers and Multiplex RT-PCR

The aim of this study was to detect the expression of 4 clinically-important efflux pumps in the Resistance-Nodulation-Cell Division (RND) family including MexAB-OprM, MexXY, MexCD-OprJ and MexEF-OprN in Pseudomonas aeruginosa using a combination of resistance-phenotypic markers and multiplex RT-PCR (mRT-PCR). The antibiotic substrates specific for each Mex systems were used as phenotypic markers including carbenicillin, MexAB-OprM, erythromycin, MexCD-OprJ, norfloxacin and imipenem, MexEF-OprN and gentamicin, MexXY-OprM. The methods were validated with reference strains with known genotypes of the Mex systems and the potential applicability in clinical practice was tested with clinical isolates. The results for the reference strains support that the combination of resistance phenotype and mRT-PCR is a potential-attractive method for diagnosis of efflux-mediated resistance in P. aeruginosa. Further development to make it more practical for clinical use and study in a larger number of clinical isolates is required.

猪主要RNA病毒病多重二温式RT-PCR检测方法的建立及初步应用

通过对Genbank登录的CSFV、PRRSV和JEV的核苷酸序列进行比对分析,找出CSFV的E2基因,PRRSV的Nsp2基因和JEV的E基因为相对保守区域[1]。利用生物学软件在保守序列分别设计一对引物,通过对PCR反应条件的优化,确定了最佳引物浓度、最佳Mg2+浓度和最佳退火温度,建立了多重二温式PCR方法检测CSFV、PRRSV和JEV。其扩增的目的片断大小分别为CSFV(482 bp)、PRRSV(576 bp)和JEV(375 bp),将传统三温式PCR过程中的退火与延伸合并为一步,从而大大节省临床检测时间,同时又能通过一个反应体系对CSFV、PRRSV及JEV三种猪主要RNA病毒病进行检测。用50份临床病料对本研究多重PCR技术和单项PCR技术进行对比验证,结果显示,两者的总符合率为93%以上。表明建立的多重PCR检测方法,具有特异、快速、准确的特点,可用于对这3种病毒的同时检测和鉴别诊断。

Multiplex Reverse Transcription-Polymerase Chain Reaction for Simultaneous Screening of 29 Chromosomal Translocation in Hematologic Malignancies

Multiplex reverse transcription-polymerase chain reaction （M-RT-PCR） has been proved to possess great clinical potential for simultaneous screening of 29 chromosomal translocations in acute leukemia. To evaluate the clinical value of M-RT-PCR in hematologic malignancies, bone marrow samples from 90 patients with various hematologic malignancies, including 25 acute myelogenous leukemia （AML）, 22 acute lymphoblastic leukemia （ALL）, 27 chronic myelogenous leukemia （CML）, 4 myeloproliferative diseases （MPD）, 3 chronic lymphoblastic leukemia （CLL）, 3 non-Hodgkin＇s lymphoma （NHL）, 3 myelodysplastic syndrome （MDS）, 2 multiple myeloma （MM） and 1 malignant histocytosis （MH） were subjected to both M-RT-PCR and chromosome karyotypic analysis. Some of cases were subjected to follow-up examination of M-RT-PCR during the period of clinical complete remission （CR） for detection of minimal residual leukemia. In our hand, 12 of 29 chromosomal translocation transcripts including TEL/PDGFR, DEK/CAN, MLL/AF6, AMLI/ETO, MLL/AF9, BCR/ABL, MLL/MLL, PML/RARα, TLS/ERG, E2A/HLF, EVⅡ and HOXⅡ were detected in 57 cases （63.3 %） of the 90 samples, which were in consistence with the results of karyotypic analysis. Furthermore, M-RT-PCR had also shown good clinical relevance when used as an approach to detect minimal residual leukemia. We concluded that M-RT-PCR could be used as an efficient and fast diagnostic tool not only in the initial diagnosis of hematologic malignancies but also in subsequent monitor of minimal residual leukemia.

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.

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.

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.

采用PCR技术检测植物病原菌

聚合酶联反应(PolymeraseChainReaction,PCR)用于各类DNA、RNA的体外扩增,具有灵敏、特异、快速等诸多优点,目前已广泛地作为人类、动植物病毒、真菌等分子检测的重要手段.并在此基础上与分子生物学其他最新技术相结合,进一步发展了IC-PCR,realtimefluorescentPCR,PCR-ELISA等复合PCR技术.将PCR技术与这些方法的突出优点相结合使用,从而提供了更快捷、特异、准确、高效检测侵染植物病原菌的方法.

Preliminary Studies on Species and Distribution of Citrus Viroids in China

Citrus viroids are the small but economically important RNA pathogens. For investigating their occurrence and distribution in China, 65 viroid samples collected from 8 major citrus cultivated regions were evaluated using one-step or multiplex onestep RT-PCR and biological indexing for specifically detection of Citrus exocortis viroid （CEVd）, Citrus bent leaf viroid （CBLVd）, Hop stunt viroid （HSVd）, Citrus viroid-Ⅲ （CVd-Ⅲ） and Citrus viroid-Ⅳ （CVd-Ⅳ）. The results showed that there were at least 4 kinds of citrus viroids （CEVd, CBLVd, HSVd, and CVd-Ⅲ） on citrus trees in China. Most of the infected citrus plants harbored more than one viroid species, and two plants were infected with up to 4 citrus viroids. Sweet orange was more frequently infected by viroids than other citrus varieties. It is the preliminary report on the species and distribution of citrus viroids in China.

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.

Identification of connexin 50 and 57 mRNA in A-type horizontal cells of the rabbit retina

Horizontal cells (HCs) mediate negative feedback to photoreceptors. In the mammalian retina, there are two types of HCs, which are extensively coupled to neighboring cells through homologous gap junctions. The permeability and therefore the strength of feedback can be regulated by light intensity, dopamine and many other factors. However, the component(s) of the most prominent gap junctions, those between A-type HCs in the rabbit retina, is still unknown. In this study, we compared the sequences of many types of mammalian connexins, obtained partial sequences of rabbit connexin 50 and 57. Using specific primers designed against the rabbit sequences, we identified mRNAs of connexin 50 and/or 57 in visually selected single A-type HC using multiplex RT-PCR.