肾综合征出血热患者血清汉坦病毒基因的PCR-ELISA方法建立

目的：建立检测肾综合征出血热（HFRS）患者血清标本中HV基因的PCR－ELISA方法。方法：设计并合成互补于HV S基因片段的标记引物。对108例HFRS患者血清进行巢式RT－PCR扩增，并用酶免方法分析扩增产物。结果：108例患者不同病日采集的血清标本的巢式RT－PCR平均检出率仅为62．0％，而PCR－ELSA平均检出率为81．5％，结论：PCR－ELISA用于早期HFRS患者的HV基因检测是一种理想的检测方法。     

荧光定量PCR检测沙眼衣原体及临床应用

目的：用荧光定量聚合酶链反应（FQ－PCR）检测沙眼衣原体的含量,探讨FQ－PCR在衣原体性尿道炎诊断中的价值。方法：应用荧光探针标记引物的荧光定量聚合酶链反应对172例疑为沙眼衣原体感染患者标本进行检测,并与常规PCR（电泳－EB染色）进行比较。结果：FQ－PCR阳性率为19．8％,常规PCR法为20．9％,两法符合率为63．9％。女性宫颈分泌物标本FQ－PCR阳性为29．2％,常规PCR法为16．7％;男性分泌物FQ－PCR阳性率为16．9％,常规PCR法为13．8％。FQ－PCR特异性较常规PCR法高。结论;FQ－PCR在扩增中实时在线检测,并选择理想的标准曲线做出较精确的临值分析,具有较高的特异性和敏感性,对病原体的诊断有一定的临床意义。     

联合应用PCR—酶切法和STR检测腓骨肌萎缩症IA型17p11.2—p12 的基因重复

目的 研究临床简化诊断腓骨肌萎缩症1A（CMT1A）型17p11.2-p12基因重复的有效方法。方法 利用聚合酶链反应（PCR）加双酶切方法,检测CMT1患者组及对照组的特异性连接片段,同时应用短串联重复序列（STR）作为遗传标记检测相同的患者,结果 22例CMT1患者中,特异性连接片段检出率为54．5％（12／22）,STR分析共检出基因重复14例,占63．6％,其它均未检测到特异性连接片段,联合应用PCR－双酶切和STR分析,基因重复出率为77．3％（17／22）,结论 PCR－双酶切法检测CMT1A基因重复具有快速,简洁,特异性好等优点,适于临床推广应用,联合应用PCR－双酶切和STR分析,可提高CMT1A患者基因重复检出率。     

重组酶聚合酶扩增结合横向流体试纸条快速检测人腺病毒CSCD

目的 建立一种快速、敏感的人腺病毒等温核酸扩增检测方法.方法 针对人腺病毒hexon基因保守区序列设计特异性重组酶聚合酶扩增（RPA）引物及探针、优化反应时间和温度、用横向流体试纸条（LFD）和毛细管电泳检测扩增产物,建立人腺病毒RPA-LFD快速检测方法,评价该方法的敏感度和特异性并与Real-time PCR法比较.结果 RPA-LFD方法检测人腺病毒的最低检出限为2拷贝DNA分子/反应,且与其他呼吸道病原无交叉反应,临床样本检测结果与Real-time PCR法一致性为100％.结论 建立的RPA-LFD方法具有敏感性、特异性高、快速且不需要昂贵的仪器设备等优点,为人腺病毒快速检测提供了新工具.     

逆转录环介导等温扩增检测风疹病毒RNA方法的建立

目的建立逆转录环介导等温扩增快速检测风疹病毒RNA的方法。方法选取保守基因E1设计引物,建立并优化RT—LAMP反应体系,同时进行特异性和敏感性评估。在此基础上应用建立的方法对46例ELISA检测阳性的孕妇血清标本进行检测,并与RT—PCR相比较。结果建立的方法仅对风疹病毒RNA扩增出特有的梯状条带,酶切结果与预期相符。46例孕妇标本经RT—PCR和RT—LAMP检测,分别检出28例和29例。结论该方法特异性强、敏感性高、简便快速且成本低,有望在临床检测中发挥一定的作用。     

特异性引物聚合酶链反应乙型肝炎病毒基因分型法的建立及应用

目的应用型特异性引物聚合酶链反应法（PCR）进行乙型肝炎病毒基因分型并分析该法的可靠性。方法应用型特异性引物PCR和INNO-LiPA分别对深圳、长春、北京152份HBV DNA阳性慢性乙型肝炎患者的血清标本进行了基因型分型，对该两种分型法不一致的血清标本再进行S区基因测序分型，以确定该两法的可靠性。结果型特异性引物PCR和INNO-LiPA的总符合率为86．8％（132／152），不一致率为13．2％（20／152）。型特异性引物PCR检测到81份（53．3％）B型；58份（38．2％）C型；13份（8．5％）B＋C型混合感染，未检出其他基因型或混合感染的基因型。INNO-LiPA检测到74份（48．7％）B型；61份（40．1％）C型；5份（3．3％）B＋C型混合感染；另检出3份（2．0％）A＋B型混合感染；1份（0．7％）B＋E型混合感染；1份（0．7％）C型与D型，1份（0．7％）D型感染，3份（2．0％）B／C／D型及3份未能分型。20份两法分型不一致的标本中，6份无剩余血清，对其余14份进行了S区基因测序分型，结果型特异性引物PCR与S区基因测序分型法的符合率为71．4％（10／14），而INNO-LiPA与S区测序法的符合率仅为7．1％（1／14），前者明显高于后者（P〈0．05）。结论型特异性引物PCR和INNO-LiPA均可鉴定HBV基因型，但前者较为简便和可靠，且费用较低，可用于临床标本的检测和流行病学调查。     

HCV E1基因序列的原核高效表达及表达产物的初步应用

目的 在原核表达系统中对HCV E1基因进行克隆和高效表达,并初步探讨表达产物在抗－HCV筛选中的作用。方法 通过RT－PCR法从HCV RNA阳性的血清标本中扩增出HCV E1片段,并在扩增用的引物中引入克隆所需的酶切位点,克隆产物经Xba I和EcoR I酶切后,在T4DNA连接酶的作用下克降人经同样双酶切的原核表达载体PMS－31b中,并用此连接产物转化大肠埃杀菌POP2136,挑取具有氨苄抗性的菌东扩大培养后提取质粒进行酶切鉴定,鉴定出的阳性菌经42℃诱导后用SDS－PAGE检查其表达状况,并用Western blot鉴定表达产物有特异性,同时用初步纯化的表达产物用ELISA法检测病人血清。结果 经Sma I酶切后PCR产物被切成144bp和356bp的两个片段,在具有氨苄抗性的菌中提取的质粒经Sma I和Xba I酶切后产生了一356bp的片段;42℃诱导后在SDS－PAGE中出现了一条相对分子质量为31000的条带,其表达量约为17％,经Western blot鉴定后在该处出现了阳性信号;ELISA实验显示在抗HCV阳性的血清中的阳性率约为29．8％（26／90）,而在抗HCV阴性的 血清中其阳性率约为3．9％（3／76）。结论 通过RT－PCR方法将HCV E1基因从HCV RNA阳性的血清标本中调出,构建了HCV E1的原核表达载体－PMS－E1,其表达量为17％,表达产物具有良好的特异性,有望应用于抗HCV的检测中。     

无菌性脑膜炎和脑炎病人脑脊液肠道病毒RNA的检测及其临床意义

目的 检测肠道病毒（EV）在中枢神经系统感染中的致病情况,探讨检测EV感染的方法。方法 就用逆转录－聚合酶链反应（RT－PCR0和病毒培技术检测46例无菌性脑膜炎及脑炎病人脑脊液（CSF）标本。结果 RT－PCR方法敏感特异;46例无菌性脑膜炎和脑炎急性期CSF标本中,31例EV阳性（67．4％）,14例病毒培养阳性（26．1％）。统计结果显示,RT－PCR敏感性明显高于病毒培养。结论 EV是引起无菌性脑膜炎和脑炎的重要病原;RT－PCR快速敏感特异,简单易行,易于推广,是诊断EV感染的有效方法。     

脑心肌炎病毒 TaqMan real-time PCR检测方法的建立及应用

目的建立脑心肌炎病毒（ EMCV） TaqMan real-time PCR检测方法。方法根据GenBank中公布的EMCV 3D基因保守区段设计并合成1对引物和1条TaqMan 探针,建立EMCV TaqMan real-time PCR检测方法,且对体系进行优化;对该法进行灵敏性、特异性验证;采用建立的方法对98份猪血清样本进行检测,并与ELISA结果进行比较。结果建立的EMCV TaqMan real-time PCR检测方法线性关系较好,以质粒标准品构建的标准曲线相关系数R2为0．995;灵敏性比普通PCR高100倍,且仅能特异性检出 EMCV;对猪血清样本的检测与 ELISA 法检测结果符合率为98．0％。结论已建立了EMCV TaqMan real-time PCR检测方法,该法灵敏性高、特异性好,可用于EMCV的检测及定量分析。     

使用PCR—SSP方法检测HLA—DR抗原

目的　采用顺序特异引物聚合酶链反应 (PCR -SSP)建立人类白细胞抗原DR位点的DNA分型方法 .方法　合成 2 9个特异性引物和 1对阳性对照引物 ,组成 2 0个PCR反应用于DR位点 ,建立一步法PCR -SSP .结果　所有样本PCR -SSP基因分型获得成功 ,分型结果经标准DNA ,限制性核酸内切酶分析证实符合 ,特异性和重复性 10 0 % .结论　PCR -SSP检测HLA -DR的方法具有快速、准确、特异性高等优点 ,适合临床应用 .     

甲型流感病毒荧光RT-PCR检测方法的设计和检定

目的　设计甲型流感病毒TaqMan荧光RT-PCR检测方法并对其进行检定。方法　用DNAStar和PrimerPremier5. 0软件设计甲型流感病毒荧光PCR检测所用引物和探针 ;在GenBank中进行Blast以及电子对比证明其具有高度的特异性和保守性 ;和标准RT-PCR进行比较 ,检测此方法的灵敏度。结果　所设计的引物和探针高度特异和保守 ,灵敏度比标准RT-PCR方法高 3～ 27倍 ,并且反转录和PCR可合并为一步。结论　设计了甲型流感病毒TaqMan检测方法 ;该方法具有特异、灵敏和简便的特点     

Genotyping of 11 human papillomaviruses by multiplex PCR with a GeXP analyzer

A new, rapid, and high-throughput method was developed for simultaneous detection of 11 human papillomavirus (HPV) genotypes including nine high-risk types (HPV16, 18, 31, 33, 35, 39, 52, 58, and 66) and two low-risk types (HPV6 and 11) in a single tube by multiplex PCR based on a GenomeLab Gene Expression Profiler (GeXP) analyzer (GeXP-PCR). Eleven sets of chimeric primers were used to initiate the PCR, and one pair of universal primers was used for the subsequent cycles of the PCR. The specificity of GeXP-PCR for each HPV type was examined with clinical samples of single type HPV infection tested previously. The sensitivity of GeXP-PCR was evaluated by performing the assay on serial 10-fold dilutions of cloned PCR products. The GeXP-PCR achieved a sensitivity of 100 copies when all of the 11 pre-mixed plasmids containing HPV targets were present. Analyses of 124 clinical specimens using the GeXP-PCR demonstrated that the GeXP-PCR assay had comparable sensitivity and specificity to those of reported multiple PCR assay and an increased detection of HPV 11 in samples with mixed infections. In conclusion, the GeXP-PCR is a fast, sensitive, and high throughput method for the detection of multiple HPV infections.     

Diagnosis of hand,foot, and mouth disease caused by EV71 and other enteroviruses by a one‐step,single tube,duplex RT‐PCR

Hand, foot, and mouth disease (HFMD) is caused mainly by enterovirus 71 (EV71) and other enteroviruses (EVs) such as Coxsackie A16 in China. EV71 infection can lead to severe clinical manifestations and even death. Other EVs, however, generally cause mild symptoms. Thus, early and accurate distinction of EV71 from other EVs for HFMD will offer significant benefits. A one‐step, single tube, duplex RT‐PCR assay is described in the present study to detect simultaneously EV71 and other EVs. The primers used for the duplex RT‐PCR underwent screening and optimization. The detection threshold was 0.001 TCID₅₀/ml for EV71 and 0.01 TCID₅₀/ml for other EVs. The positive rate of enterovirus detection in 165 clinical samples reached 68.5%, including 46.1% for EV71 and 22.4% for other EVs. Of all the severe HFMD cases, EV71 was responsible for 85.3% cases. The positive rate of EV71 fell markedly by day 8 after onset. In addition, sequencing of EV71 specific amplicons from duplex RT‐PCR revealed that C4a was the predominant subgenotype of EV71 circulating in Nanjing, China. The accuracy and reliability of the assay suggest strongly that the one‐step, single tube, duplex RT‐PCR will be useful for early diagnosis and monitoring of EV71 and other EV infections. J. Med. Virol. 84:1803–1808, 2012. © 2012 Wiley Periodicals, Inc.     

Development and evaluation of one‐step multiplex real‐time RT‐PCR assay for simultaneous detection of Zika virus and Chikungunya virus

Zika virus (ZIKV) and chikungunya virus (CHIKV) are important human pathogens and mosquito‐borne arboviruses, which have resembling history, common vectors, circulating regions, and indistinguishable clinical symptoms. Wide geographical range that is suitable for ZIKV and CHIKV transmission underlines the concern about the impact of epidemic and endemic infections on burden of public health. In the present study, a highly sensitive and specific one‐step multiplex real‐time RT‐PCR assay was developed and evaluated for simultaneous detection and quantification of ZIKV and CHIKV. The single reaction assay employs two pairs of primers and two TaqMan probes that differentiate ZIKV and CHIKV infections. The entire viral genomic RNA in vitro transcribed from full‐length infectious clones were used to generate the standard curves for absolute quantification in subsequent tests. The detection limit of the one‐step multiplex assay was 1 and 0.5 PFU for infectious ZIKV and CHIKV, respectively. The assessment of specificity indicated this assay is highly specific to targeted viruses showing no amplification of a variety of other flaviviruses. Our assay was able to detect geographically separated and phylogenetically diverse strains of ZIKV and CHIKV. On the applicability of monitoring viral multiplication in cells and testing clinical samples, the one‐step multiplex assay provided efficient and accurate determination. The one‐step multiplex real‐time RT‐PCR assay offers a valuable tool for detection of ZIKV and CHIKV and potentially contributes to general surveillance and clinical treatment.

     

Diagnosis and differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36 and p46 genes

The genome of Mycoplasma hyopneumoniae encodes several immunodominant proteins, including a cytosolic protein (p36), three membranous proteins (p46, p65, and p74), and an adhesin (p97). Cross-reactions with M. flocculare and M. hyorhinis reduce the specificity of conventional serological detection methods. However, certain antigenic determinants of the p36 and p46 proteins have been shown to be specific for M. hyopneumoniae. In the present study, pairs of oligonucleotide primers were designed to permit PCR amplification of entire p36 and p46 genes and of internal fragments of these genes. Specific amplicons could be obtained with as low as 0.5 to 50 pg of extracted chromosomal DNA. No amplification product was obtained when testing p36 and p46 primer pairs with genomic DNA or RNA from other mycoplasma species, bacteria, and viruses commonly associated with respiratory diseases in pigs. By using the single p36-PCR method, a positive reaction was demonstrated in 100% (30 of 30) of lungs from pigs that developed typical lesions associated with an M. hyopneumoniae infection, and no false-positive results were detected when 62 apparently normal lungs were tested. On the other hand, with the single p46-PCR method a sensitivity of 86.6% (26 of 30) and a specificity of 96.7% (60 of 62) were obtained in comparison with the necropsy findings. A mixed infection with M. hyorhinis was diagnosed in 13.3% (4 of 30) of the cases by using species-specific primers for the heterologous p37 gene. The sensitivity of the single p36-PCR method for the detection of M. hyopneumoniae, when tested on tracheobronchial swabs, was 100% (20 positive samples), with a specificity of 93.3% (14 of 15 negative samples), compared to the necropsy findings. Both expected amplicons were obtained with 86.6% (26 of 30) positive lungs when p36 and p46 primers were used simultaneously (multiplex PCR) to further increase the specificity of the PCR assay.     

PCR strategy for identification and differentiation of small pox and other orthopoxviruses.

Rapid identification and differentiation of orthopoxviruses by PCR were achieved with primers based on genome sequences encoding the hemagglutinin (HA) protein, an infected-cell membrane antigen that distinguishes orthopoxviruses from other poxvirus genera. The initial identification step used a primer pair of consensus sequences for amplifying an HA DNA fragment from the three known North American orthopoxviruses (raccoonpox, skunkpox, and volepox viruses), and a second pair for amplifying virtually the entire HA open reading frame of the Eurasian-African orthopoxviruses (variola, vaccinia, cowpox, monkeypox, camelpox, ectromelia, and gerbilpox viruses). RsaI digest electropherograms of the amplified DNAs of the former subgroup provided species differentiation, and TaqI digests differentiated the Eurasian-African orthopoxviruses, including vaccinia virus from the vaccinia virus subspecies buffalopox virus. Endonuclease HhaI digest patterns distinguished smallpox variola major viruses from alastrim variola minor viruses. For the Eurasian-African orthopoxviruses, a confirmatory step that used a set of higher-sequence-homology primers was developed to provide sensitivity to discern individual virus HA DNAs from cross-contaminated orthopoxvirus DNA samples; TaqI and HhaI digestions of the individual amplified HA DNAs confirmed virus identity. Finally, a set of primers and modified PCR conditions were developed on the basis of base sequence differences within the HA genes of the 10 species, which enabled production of a single DNA fragment of a particular size that indicated the specific species.     

Sequencing-based typing of HLA-A locus using mRNA and a single locus-specific PCR followed by cycle-sequencing with AmpliTaq DNA polymerase, FS

The large number (59) of alleles now known at the HLA-A locus is a serious challenge to the existing methods for HLA typing, including many of the DNA based methods. Here, we describe a sequencing-based typing (SBT) protocol for typing of HLA-A alleles using a single A-locus-specific PCR. This reaction amplifies an 824 base pair product from cDNA, prepared from mRNA, covering exons 1–3 and most of exon 4. This product allows identification of all possible combinations of two alleles from this locus. The sequencing strategy used for allele assignment contains several improvements compared to those previously published. The enzyme AmpliTaq® DNA Polymerase, FS, used, combines high-quality sequencing, i.e. long reads, low background, and uniform peak heights making the identification of heterozygous positions very reliable in a fast and easy protocol developed by determining the optima for a number of variables. Thus, this strategy meets most of the requirements for the use of sequencing in HLA typing. Furthermore, this method is very flexible. The use of a PCR primerpair tailed with the recognition sites for two different sequencing primers allows the application of the same sets of fluorescent-labelled sequencing primers regardless of the amplified locus. Thus, the protocol can very easily be extended to cover the B- and C-locus too, simply by adding PCR reactions specific for these loci to the protocol. Using this protocol, we investigated a total of 65 cell lines and clinical samples, many of the latter chosen from samples difficult to type by serology. Our method gave in all cases unambiguous results and proved functional for work requiring the highest resolution.