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.

Development and Validation of Multiplex One-Step Real-Time TaqManqRT-PCR Assays for Detection and Quantification of Arboviral Encephalitis Viruses

Arboviral encephalitis is a group of animal and human illness that is mostly caused by several distinct families of viruses including orthobunya virus, phlebovirus, flaviviruses, and the alphaviruses. Although specific signs and symptoms vary by the type of central nervous system (CNS), initial signs and symptoms are very similar. Therefore rapid immunologic and molecular tools for differential diagnosis of arboviral encephalitis viruses are important for effective case management and control of the spread of encephalitis. The qRT-PCR assay, especially multiplex PCR, has the potential to produce considerable savings in time and resources in the laboratory detection. Meanwhile, the use of IC can prevent false negatives effectively by monitoring the processes of nucleic acid extraction and amplification. This report describes the development of a panel of internally controlled multiplex one-step real-time RT-PCR assays in which two virus specific-probe sets were used in the same reaction for the detection of 15 species arboviral encephalitis viruses: the comparative sensitivity of multiplex one-step qRT-PCR assays to single plex one-step qRT-PCR assays as well as one-step RT-PCR assays for detection of each viral species. And total of 150 human serum samples were detected to evaluate the multiplex one-step qRT-PCR assays. These multiplex one-step real-time RT-PCR assays with IC were evaluated in terms of sensitivity, linearity, precision, specificity, and also field samples including serum and vector. These assays can detect and differentiate arboviral encephalitis viruses by high throughput, sensitive, and specific way. It is useful for clinical management and outbreak control of arboviral encephalitis viruses and vector surveillance.

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.

One-Step Multiplex PCR for Simultaneous Detection and Identification of Eight Medically Important <i>Candida</i>Species

Recently, the incidence of Candida infections has substantially increased. Conventional identification methods for Candida species are technically difficult to conduct and cannot accurately distinguish each species. The purpose of the present study was to design primers to identify and detect simultaneously eight medically important Candida species using one-step multiplex PCR. PCR primers were designed based on partial sequences of intergenic spacer (IGS) and internal transcribed spacer (ITS) genes of eight medically important Candida species. These primers were able to distinguish each Candida species and did not display cross-reactivity with representative Candida species other than the eight Candida species. Moreover, our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

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.

One Step Multiplex PCR for Identifications at Subspecies Level of Fusobacterium nucleatum and Fusobacterium necrophorum

Purpose: Fusobacterium nucleatum is an opportunistic pathogen involved in periodontal diseases, extraoral infections, and colorectal cancer. Fusobacterium necrophorum causes a variety of necrotic infections. F. nucleatum and F. necrophorum are classified into five and two subspecies, respectively. Conventional identification methods were technically hard to distinguish each subspecies of two Fusobacterium species accurately. The purpose of the present study was to design primers to identify two medically important Fusobacterium species at the subspecies level, using one-step multiplex PCR. Methods: Polymerase chain reaction (PCR) primers were designed based on partial sequences of the 16S ribosomal RNA (16S rRNA) gene, RNA polymerase B (rpoB) gene, and DNA gyrase subunit B (gyrB) of each subspecies of F. nucleatum and F. necrophorum. Results: These primers were able to distinguish each subspecies of F. nucleatum and F. necrophorum and did not display cross-reactivity with representative Fusobacterium species other than F. nucleatum and F. necrophorum. Conclusion: Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction.

两步多重RT-PCR快速检测苹果潜隐性病毒

【目的】为建立检测3种苹果潜隐性病毒更为快速准确的方法，【方法】以携带苹果茎沟病毒（ASGV）、苹果褪绿叶斑病毒（ACLSV）和苹果茎痘病毒（ASPV）的成龄苹果树韧皮部为试材，根据基因库中苹果茎痘病毒（Apple stempitting virus，ASPV）的外壳蛋白基因序列，设计合成了2对特异性引物，分别与合成的苹果茎沟病毒（ASGV）引物和苹果褪绿叶斑病毒（ACLSV）的引物组合配伍，筛选出最佳的引物对组合。对eDNA的合成所用引物和总RNA模板进行遴选和量化，对PCR过程中eDNA的用量、引物对的终浓度、退火温度等主要影响因素进行优化。【结果】结果表明，反转录引物为Oligo（dT）18、总RNA0．1～3．0μL时，可以得到较好的eDNA；ASPV—FR与ASGV—Pch、ACLSV—Pch引物组合优于ASPV—Pch，并且筛选出4组最佳的终浓度处理组合；eDNA用量为2．0-4．0μL、退火温度为50．0～52．9℃、循环数为35时，扩增效果较好。采用优化的多重RT—PCR体系对采自陕西和山东两省的样品进行检测，并用3种病毒的单重RT—PCR体系进行验证。【结论】结果呈现高度一致性，充分印证了该多重RT—PCR体系的准确性，适用于大量样品的快速检测。

一步法多重荧光定量RT-PCR检测人杯状病毒的建立及应用

目的建立检测人杯状病毒(human calicivirus,HuCVs,主要包括SV、NoV-GⅠ和NoV-GⅡ)的一步法多重荧光定量RTPCR(rRT-PCR)法。方法用Primer Express 3.0软件设计特异性引物和探针,通过合成的对应病毒基因的质粒来评价该方法的灵敏度、特异性和重复性,检测临床标本并对阳性结果进行基因测序验证。结果建立的rRT-PCR法检测SV、NoV-GⅠ和NoV-GⅡ的灵敏度均达到103copies/mL,特异性为100%,且变异系数(CV)均≤1.275%。HuCVs检出率为21.43%(75/350),其中SV、NoV-GⅠ和NoV-GⅡ分别为14.67%(11/75)、21.33%(16/75)和64.00%(48/75)。随机选择检测阳性结果进行测序,结果均与该法检测结果一致。结论建立的rRT-PCR法可同时检测并区分SV、NoV-GⅠ和NoV-GⅡ病毒,可用于HuCVs引起的急性腹泻散发和暴发疫情的监测。

一步法多重RT-PCR检测猪圆环病毒2型和猪繁殖与呼吸道综合征病毒

根据猪圆环病毒2型(PCV2)的ORF2和猪繁殖与呼吸道综合征病毒(PRRSV)的ORF7相对保守区序列分别设计了两对引物PH1/PH2和PT1/PT2,将PCV2和PRRSV细胞毒按1:1混合,设为模拟样品,采用病毒基因组DNA/RNA提取试剂盒同时提取PCV2DNA和PRRSV RNA核酸,利用2对引物进行一步法多重RT-PCR。结果同时得到与实验设计相符的560bp(PCV2)和398bp(PRRSV)特异性扩增条带,而对其他5种猪病原的扩增均为阴性。敏感性试验表明,建立的一步法多重RT-PCR方法可检测出10ngPCV2DNA和5ngPRRSV RNA。

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。

猪繁殖与呼吸综合征病毒一步多重RT-PCR检测方法的建立及应用

本研究旨在建立猪繁殖与呼吸综合征病毒(PRRSV)的一步多重RT-PCR检测方法。通过多序列比对以及反应条件的优化,从4对引物中精选了3对特异性和敏感性较好、扩增片段分别为228、345和490bp的引物,并应用所建立的方法对4种分离株的细胞毒和组织毒、田间样品及临床症状相似的疾病猪瘟和猪伪狂犬病毒做了检测;并对田间及实验室样品进行符合率试验,对阳性样品进行重复性试验。与常规RT-PCR法进行比较,证明该方法比常规RT-PCR法敏感性提高了10倍;而且利用该方法可使检测时间大大缩短;特异性试验证实该方法无交叉反应;阳性和阴性样本的符合率均为100%;3次阳性样品重复性检测结果完全相同;稳定性试验显示该试剂盒至少可保存1年以上。本研究所建立的PRRSV一步多重RT-PCR检测方法的敏感性高、特异性强、操作简便、省时、结果重复性好,不仅适用于临床病例尤其低微含量样品的诊断,而且在筛查隐形带毒动物及兽医检疫方面具有重要的应用价值。

3种苹果潜隐性病毒一步多重RT-PCR检测体系的优化

为建立更为快速准确的苹果潜隐性病毒检测方法,以携带苹果茎沟病毒(ASGV)、苹果褪绿叶斑病毒(ACLSV)和苹果茎痘病毒(ASPV)的田间苹果成龄树为试材,设计合成了扩增目的片段为273bp(ASGV)、358bp(ACLSV)和432bp(ASPV)的3对特异性引物,并对影响RT-PCR体系的主要参数进行试验优化。结果表明:建立的一步多重RT-PCR体系可以实现对3种主要潜隐性病毒的特异性检测;通过对田间多个样品的检测验证,表明该方法的准确性和灵敏度都很高,并缩短了单个样品的检测时间,极大地提高了工作效率。

多重RT-PCR一步法技术同时检测猪瘟病毒和蓝耳病病毒方法的建立以及初步应用

猪瘟病毒和蓝耳病病毒均能导致猪繁殖障碍,对养猪生产影响很大。根据猪瘟病毒(CSFV)和猪蓝耳病(PRRS)的基因保守序列设计2对针对这2种病毒的特异引物,并建立多重RT-PCR方法,分别对其最佳反应条件、特异性及敏感性进行测定。结果表明,能同时扩增得到2条与试验设计相符的167bp(CSFV)和320bp(PRRS)特异性条带,同时具有较好的特异性;敏感性检测结果表明,临床阳性的样品提取的核酸稀释1000倍后仍能检测出CSFV和PRRSV。本方法的建立对于这2种病毒病的早期快速检测具有十分重要的意义。

面向小型无人机的OFDM-MIMO雷达信号设计和处理方法

随着空域的逐渐开放,以小型无人机为代表的低慢小目标,其有效检测对维护安全非常重要。然而,低慢小目标具有飞行高度低、飞行速度慢及雷达散射截面积(Radar Cross Section,RCS)小的特点,加之电磁环境复杂多变,使其难以被传统低分辨雷达发现。为了提升雷达的低截获性能和成像分辨率,本文提出了一种基于步进正交频分复用(Orthogonal Frequency Division Multiplexing,OFDM)的多输入多输出(Multiple Input Multiple Output,MIMO)雷达信号设计和处理方法。该方法是将窄带OFDM信号与非线性步进方案相结合,通过非线性步进方案控制MIMO发射信号的载频,获得发射端正交波形和大射频带宽,使得雷达接收机的天线孔径获得明显扩展,从而提升雷达的低截获性能和角度分辨率,同时实现低采样速率下的高距离分辨率。在雷达接收端,利用发射波形之间的正交性分离各通道回波,然后基于改进离散傅里叶变换(Discrete FourierTransform,DFT)和解码的处理方法,校正了非线性步进方案产生的相位误差,生成目标的三维高分辨成像。本文在77 GHz载频下设计仿真,验证所提方法的有效性。仿真结果表明,所提方法相比现有方法,在低信噪比下具有较高的三维成像分辨率,而且没有明显增加信号处理的复杂度。然而,该方法减小了最大无模糊速度。此外,本文讨论了步长数对雷达性能的影响,仿真结果表明,随着步长数的增加,最大无模糊速度减小,角度分辨率提升,为实际应用场景下步进OFDM-MIMO雷达的信号设计提供理论指导。

一步法多重RT-PCR检测猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪A群轮状病毒方法的建立和应用

根椐Gen Bank中登录的猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪A群轮状病毒(PARV)基因序列,分别设计3对引物,在建立各病毒单项一步法RT-PCR技术的基础上,优化多重RT-PCR反应条件,建立了3种病毒的一步法多重RT-PCR检测方法,用这3对引物对同一样品中的PEDV、TGEV和PARV核酸模板进行多重RT-PCR扩增,结果可同时扩增PEDV的450 bp、TGEV的609 bp、PARV的241 bp特异性片段,而对其它4种病原的扩增结果均为阴性。敏感性测定结果表明,该多重RT-PCR技术能检出10 pg的PEDV、10 pg的TGEV和1 pg的PARV模板。用67份临床病料对建立的多重RT-PCR技术和单项RT-PCR技术进行对比验证,结果显示,两者的总符合率为100%。表明建立的一步法多重RT-PCR检测方法,具有特异、快速、准确的特点,可用于这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.