Nested RT-PCR method for the detection of European avian-like H1 swine influenza A virus

Swine influenza A virus（swine IAV） circulates worldwide in pigs and poses a serious public health threat, as evidenced by the 2009 H1N1 influenza pandemic. Among multiple subtypes/lineages of swine influenza A viruses, European avian-like（EA） H1N1 swine IAV has been dominant since 2005 in China and caused infections in humans in 2010. Highly sensitive and specific methods of detection are required to differentiate EA H1N1 swine IAVs from viruses belonging to other lineages and subtypes. In this study, a nested reverse transcription（RT）-PCR assay was developed to detect EA H1 swine IAVs. Two primer sets（outer and inner） were designed specifically to target the viral hemagglutinin genes. Specific PCR products were obtained from all tested EA H1N1 swine IAV isolates, but not from other lineages of H1 swine IAVs, other subtypes of swine IAVs, or other infectious swine viruses. The sensitivity of the nested RT-PCR was improved to 1 plaque forming unit（PFU） m L^（-1） which was over 10~4 PFU m L^（-1） for a previously established multiplex RT-PCR method. The nested RT-PCR results obtained from screening 365 clinical samples were consistent with those obtained using conventional virus isolation methods combined with sequencing. Thus, the nested RT-PCR assay reported herein is more sensitive and suitable for the diagnosis of clinical infections and surveillance of EA H1 swine IAVs in pigs and humans.

Nested multiplex PCR for identification and detection of human Plasmodium species including Plasmodium knowlesi

Objective:To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria.Methods:In this study the nested multiplex malaria PCR was redesigned,targeting the 18S rR NA gene,to identify the fifth human Plasmodium species,Plasmodium knowlesi,together with the other human Plasmodium(Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale and Plasmodium malariae)by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives.Results:The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms.The technique showed high sensitivity(100%)and specificity(96%)when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos栿and a published real-time PCR malaria assay.Conclusions:The technique designed is an economical,sensitive and specific alternative to current diagnosis methods.Furthermore,the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.

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.

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.

Detection of EWS-FLI1 fusion transcripts in paraffin embedded tissues of peripheral primitive neuroectodermal tumors by nested reverse transcription polymerase chain reaction

Objective: To assess the feasibility and significance of detecting EWS-FLI1fusion transcripts in paraffin embedded tissues of peripheral primitive neuroectodermal tumors (PNETs) by nested reverse transcription polymerase chain reaction(RT-PCR). Methods: Twelve formalin-fixed and paraffin-embedded (FFPE) samples of PNET were retrieved from archive and consultation materials, together with eight cases of controlled tumor. EWS-FLI1 fusion transcripts were detected by nested RT-PCR. Home-keeping gene β-actin was used to detect the quality of mRNA. Results: β-actin mRNA was detected in 9 of the 12 tumor cases. EWS-FLI1 fusion transcripts were detected in 6 cases, among which 4 had a “type 1” fusion transcript and 2 had a “type 2” fusion transcript. None of the controlled tumor was detected the fusion gene. Conclusion: RT-PCR is a feasible method for the detection of EWS-FLI1 fusion transcripts in FFPE tissues in PNET and the result is meaningful in differential diagnosis and prognostic evaluation.

多重套式RT-PCR检测患者凝血块中登革病毒RNA(英文)

目的建立简便、灵敏、适合于全部血清型登革病毒核酸检测的多重套式RT-PCR体系,检测临床样品中登革病毒RNA,作为实验室辅助诊断的依据。方法利用登革病毒标准株核酸,建立多重RT-PCR检测方法。提取患者凝血块总RNA,分别用一步法RT-PCR及多重套式RT-PCR检测。结果通过对检测体系进行优化,多重RT-PCR能够同时检测4种血清型登革病毒核酸。采用多重套式RT-PCR方法,从8例登革热患者凝血块中有4例检测到病毒RNA,而其它核酸检测方法仅检出1例阳性。结论多重套式RT-PCR的方法能够从临床凝血块样品中检测到登革病毒核酸,并同时进行血清学分型,简化了登革病毒核酸检测步骤,有利于对临床样品开展病毒核酸检测。

多重巢式RT-PCR对骨髓增殖性肿瘤中PDGFRα融合基因的快速检测

本研究旨在探讨多重巢式RT-PCR在骨髓增殖性肿瘤(MPN)中快速检测血小板衍生生长因子受体α(platelet-derived growth factor receptor alpha,PDGFRα)融合基因的应用价值。利用多重巢式RT-PCR方法对146例MPN患者的骨髓或外周血标本进行PDGFRα融合基因的快速检测。结果表明,146例MPN患者的骨髓或外周血标本中,有6例出现PDGFRα融合基因,其阳性率为4.11%,其中4例患者服用酪氨酸激酶抑制剂伊马替尼治疗,均获疗效。结论:本研究建立的多重巢式RT-PCR方法具有灵敏度高、特异性好、检测快速等优点,可以准确快速确定MPN的分子类型,并为该病提供诊断和治疗的理论依据。

应用多重套式RT-PCR检测儿童急性淋巴细胞白血病的融合基因

目的 研究染色体结构易位所致的融合基因在儿童急性淋巴细胞白血病 (ALL)的表达。方法 采用多重套式RT PCR结合染色体R带或G带核型分析、流式细胞仪细胞免疫表型检测对 64例儿童ALL进行分析。结果 64例ALL患儿中 23例(36. 0% )具有 13种染色体畸变产生的融合基因,包括E2A/PBX1,E2A/HLF,TEL/AML1,TLS/ERG,MLL/AF4,MLL/AF9,MLL/AF10,MLL/AFX,MLL/AF6,MLL/ELL,dupMLL,TAL1D,HOX11。ALL患儿融合基因和染色体总畸变率为 67. 2%(43 /64)。结论 多重套式RT PCR结合染色体核型、免疫表型是儿童ALL临床诊断、治疗和预后判断的重要依据。

应用多重巢式RT-PCR检测骨髓增生异常综合征中MLL基因相关的10种融合基因

本研究探讨应用多重巢式逆转录聚合酶链反应(RT-PCR)检测骨髓增生异常综合征MDS)患者MLL基因相关的融合基因的临床价值。采用多重巢式RT-PCR方法检测了221例MDS患者10种MLL基因相关融合基因(dupMLL、MLL-ELL、MLL-ENL、MLL-AF6、MLL-AF9、MLL-AF10、MLL-AF17、MLL-CBP、MLL-AF1P、MLL-AF1Q)。结果表明:在221例MDS患者中检测出以上融合基因者20例(9.05%),以上10种基因的阳性例数及阳性率分别依次为7(3.16%)、2(0.9%)、1(0.45%)、1(0.45%)、2(0.9%)、2(0.9%)、1(0.45%)、2(0.9%)、1(0.45%)、1(10.45%)。结论:多重巢式RT-PCR技术能同时检测MDS患者中的10种融合基因,可作为MDS诊断及疗效判定的重要依据,同时也为微小残留病(MRD)及预后提供相关的重要信息。

多重RT-PCR法检测燕窝及其制品掺伪成分

目的建立一种食用燕窝中主成分及掺伪成分的双重和三重实时荧光PCR检测方法。方法本研究通过合成金丝燕成分、银耳成分、红藻成分特异性的引物和探针,建立燕窝中主成分及掺伪成分的双重和三重实时荧光PCR检测体系,并设计相应的特异性实验保证检测体系的准确性,最后使用所建立的方法对市售样品进行检测并收集数据,验证多重实时荧光PCR检测方法的适用性。结果本方法可在1 d内完成样品的检测,建立的体系对金丝燕成分、银耳成分的检测灵敏度为0.1 ng/μL,红藻成分的检测灵敏度为0.5 ng/μL;燕窝中掺入银耳的检测限可达到0.1%;燕窝中掺入石花菜的检测限可达到0.5%。结论本研究建立的多重实时荧光PCR检测方法具有准确、灵敏度高、特异性好的优点,可用于燕窝及其制品中掺假物成分的准确定性检测。

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.

Nested multiplex PCR——A feasible technique to study partial community of arbuscular mycorrhizal fungi in field-growing plant root

Plant can be infected by different arbuscular mycorrhizal fungi, but little is known about the interaction between them within root tissues mainly because different species cannot be distin-guished on the basis of fungal structure. Accurate species identification of Arbuscular mycorrhizal fungi (AMF) colonized in plant roots is the cornerstone of mycorrhizal study, yet this fundamental step is impossible through its morphological character alone. For accurate, rapid and inexpensive detec-tion of partial mycorrhizal fungal community in plant roots, a nested multiplex polymerase chain reac-tion (PCR) was developed in this study. Five discriminating primers designed based on the variable region of the 5′ end of the large ribosomal subunit were used in the experiment for testing their speci-ficity and the sensitivity in nested PCR by using spores from Glomus mosseae (BEG12), Glomus intraradices (BEG141), Scutellospora castaneae (BEG1) and two unidentified Glomus sp. HAUO3 and HAUO4. The feasibility assay of nested multiplex PCR was conducted by use of spore mixture, Astragalus sinicum roots co-inoculated with 4 species of arbuscular mycorrhizal fungi from pot cul-tures and 15 different field-growing plant roots respectively after analyses of the compatibility of primers. The result indicated that the sensitivity was in the same range as that of the corresponding single PCR reaction. Overall accuracy was 95%. The efficiency and sensitivity of this multiplex PCR procedure provided a rapid and easy way to simultaneously detect several of arbuscular mycorrhizal fungal species in a same plant root system.

Diagnosis of the accurate genotype of HKαα carriers in patients with thalassemia using multiplex ligation-dependent probe amplification combined with nested polymerase chain reaction

Background:Patients carrying the HongKongαα(HKαα)allele and-α3.7/αααanti-4.2 could be misdiagnosed as-α3.7/ααby the current conventional thalassemia detection methods,leading to inaccurate genetic counseling and an incorrect prenatal diagnosis.This study was aimed to accurately analyze the genotypes of HKααcarriers and-α3.7/αααanti-4.2.Methods::Samples were collected in our hospital from July 2017 to October 2019.Twenty-four common types of Chinese thalassemia were screened by gap-polymerase chain reaction(Gap-PCR)and reverse dot blot(RDB).Anti-4.2 multiplex-PCR was used to confirm carriers of theαααanti-4.2 duplication with-α3.7 deletion.Two-round nested PCR and multiplex ligation-dependent probe amplification(MLPA)were applied to accurately identify and confirm their genotypes.For data analysis,we used descriptive statistics and Fisher’s exact tests.Results::Two thousand five hundred and forty-four cases were identified as thalassemia in 5488 peripheral blood samples.The results showed thatα,β,andαβcompound thalassemia were identified in 1190(46.78%),1286(50.55%),and 68(2.67%)cases,respectively.A total of 227 samples from thalassemia patients were identified as-α3.7/ααby Gap-PCR,and the genotypes of two samples were uncertain.There was a difference between Gap-PCR and combined groups(Gap-PCR combined with nested PCR and MLPA)in detecting HKαα(P<0.05).Among the 229 patients,20 patients were identified as HKααcarriers and one was identified as-α3.7/ααα anti-4.2 by two-round nested PCR and MLPA,including 15 patients with HKαα/αα,three with HKαα/αα and β-thalassemia coinheritance,one with HKαα/-SEA,one with HKαα/-α4.2 andβ-thalassemia coinheritance,and one with-α3.7/αααanti-4.2 and β-thalassemia coinheritance.Conclusions::αααanti-4.2 and HKααgenotypes of patients carrying-α3.7 need to be detected to reduce the misdiagnosis rate of patients carrying HKααand-α3.7/αααanti-4.2 alleles.More accurate genetic counseling can be provided in the clinic using nested PCR combined with MLPA.

浙贝母两种主要病害病原菌巢式多重PCR检测方法的建立

浙贝母Fritillaria thunbergii炭疽病与干腐病作为浙贝母上常见的病害,严重影响浙贝母的质量和产量。因此,亟需建立一种快速准确的检测手段,这对病害早期诊断、早发现、早防治具有重要意义。本文以宁波市海曙区章水镇的浙贝母为材料,在前期明确引起上述两种真菌病害的病原菌分别为细线炭疽菌Colletotrichum lineola和尖孢镰刀菌Fusarium oxysporum的基础上,根据ITS序列,分别设计了两对特异性引物FO1-F/FO1-R和BMTJ4-F/BMTJ4-R,建立了针对浙贝母炭疽病与干腐病的巢式多重PCR检测方法。试验结果表明,建立的巢式多重PCR检测体系特异性好,同时检测2种病原菌的灵敏度高达100pg DNA/μL,对10株田间患病的浙贝母植株进行检测,4个样品检测出细线炭疽菌,2个样品检测出尖孢镰刀菌,另有4个样品同时检测出2种病原菌,检出率高达100%,表明该方法可以用于浙贝母炭疽病与干腐病的鉴定,且快速、准确、灵敏,研究结果为浙贝母炭疽病与干腐病的田间检测提供技术支持。

中缅边境(西段)传疟媒介的初步调查

目的了解中缅边境(西段)传疟媒介的分布与构成。方法 2008年8～9月,在中缅边境的中国云南省盈江县及其相邻的缅甸昔懂县6个自然村,用诱蚊灯在人房和牛棚共进行20次通宵诱捕。将捕获的蚊虫以传统方法进行形态学鉴定,然后用复合PCR法鉴别微小按蚊、乌头按蚊和杰普按蚊。同时,抽提部分蚊虫标本总基因组DNA,以巢式PCR方法检测蚊体内的疟原虫感染情况。结果共捕获各类蚊虫4 571只,隶属9属50种,其中按蚊属是优势蚊种,占总量的54.32%(2 483/4 571)。人房和牛棚的按蚊蚊种构成差异有统计学意义,其中人房以腹簇按蚊、微小按蚊和中华按蚊为主,而牛棚以腹簇按蚊(223只)、环纹按蚊(184只)、迷走按蚊(131只)和杰普按蚊(129只)为主。对比有牛村和无牛村中人房的蚊种构成发现,有牛村的人房以微小按蚊(260只)和腹簇按蚊(49只)为主,而无牛村人房则以腹簇按蚊(481只)和中华按蚊(124只)为主。巢式PCR检测1 075只按蚊,其中9只检出疟原虫阳性,分别为微小按蚊(7/408)、乌头按蚊(1/125)和伪威氏按蚊(1/101)。经测序鉴定均为恶性疟原虫感染,目的条带长204 bp。结论中缅边境(西段)蚊虫密度高、种类多,在传疟作用中以微小按蚊最为重要,乌头按蚊和伪威氏按蚊亦为当地的传播媒介。

单增李斯特菌不同PCR快速检测方法比较

目的比较3种PCR快速检测方法在检测单核细胞增生李斯特菌时的特异性和灵敏度方面的差异。方法针对单核细胞增生李斯特菌的毒力基因iap和prfA基因,分别设计引物,进行单个PCR、多重PCR、套式PCR等3种方法检测。结果3种PCR方法均具有较强的特异性;套式PCR的灵敏度为102cfu/ml,高于单个PCR(103cfu/ml)和多重PCR(104cfu/ml)。结论多重PCR在特异性检测方面具有优势,而在灵敏度方面,套式PCR要明显优于单个及多重PCR方法。