解脲脲原体套式（Nested）PCR检测研究

本文报告解脲脲原体（ＵＵ）的套式（ＮＥＳＴＥＤ）ＰＣＲ检测方法。经方法学考核表明，本法的特异性、灵敏度以及试剂的稳定性和对临检标本的顺应性均较好。９３份各种生殖道炎症患者之宫颈拭子标本套式ＰＣＲ检出２１份阳性（阳性率２２．６％），而市售ＰＣＲ试剂盒仅检出１份阳性（阳性率１．１％）。前者阳性检出率明显高于后者（Ｐ＜０．０１）。     

利用多组套式引物检测庚型肝炎病毒RNA

利用庚型肝炎病毒（ＧＢＶ－Ｃ／ＨＧＶ）ＮＳ３～５区序列合成了四组套式引物，建立了灵敏、特异的庚型肝炎病毒ＲＮＡ双扩增聚合酶链反应（ＰＣＲ）检测方法。用此方法检测了１０份庚型肝炎病毒抗体阳性患者血清及１０份阴性的健康人血清。前者不同组引物的检出率为ＮＳ３（１）引物９份阳性，ＮＳ３（２）引物８份阳性，ＮＳ４引物４份阳性，ＮＳ５（１）引物５份阳性，ＮＳ５（２）引物９份阳性；后者各组引物检测均为阴性。结果表明，庚型肝炎病毒不同区域引物用于ＲＴ－ＰＣＲ检出率差异较大。ＮＳ３（１）及ＮＳ５（２）这两组引物检出率最高，更适合用于ＲＴ－ＰＣＲ检测庚型肝炎病毒ＲＮＡ。     

新生儿包涵体结膜炎CT-nPCR检测的分析

本文对８５例新生儿结膜炎患儿进行沙眼衣原体套式ＰＣＲ（ＣＴ－ｎｅｓｔｅｄＰＣＲ）进行检测。２１例ＣＴ检测阳性（２４．９％）， 时对其母亲生殖道分泌物也做了检测，结果均为ＣＴ阳性。其中２０例为阴道分娩，１例剖宫产。说明新生儿包涵体结膜炎为母婴传播性疾病。孕期筛查ＣＴ，并给以有效治疗，减少新生儿患病率，达到优生优育的目的。     

常熟地区育龄妇女生殖道炎症者UNC感染的套式PCR检测

ＮＵＣ病原体已是我国育龄妇女生殖道炎症最常见的病原体群。ＮＵＣ感染并可致不良妊娠结局。为了解江苏常熟地区育龄妇女生殖道炎症者的ＮＵＣ感染状况。我们应有 式ＰＣＲ（ｎＰＣＲ）法检测了患者宫颈脱落细胞中的ＮＧ、ＵＵ、ＣＴ的特异性ＤＮＡ。结果：①ＮＵＣ ｎＰＣＲ检测特异性、正确性较好;②本地区育龄妇女生殖道炎症者中ＮＧ、ＵＵ、ＣＴ的阳性检出率分别高达２８．２％、２６．７％、１３．８％。本文对ＮＵＣ感染的检测方法和危害性进行了讨论。     

套式PCR鉴定精液解脲脲原体培养结果报告

应用套式（Ｎｅｓｔｅｄ）ＰＣＲ鉴定７０例精液解脲脲原体培养结果。显示两种方法的阳性符合率为７８．０４％，其中包括：１．培养结果２４－４８小时阳性，而后转阴性１３例，经套式ＰＣＲ检测６例阳性，阳性率为４６．１％，占阳性经率的１６．２％（６／３７）。２．培养７２小时后才显示阳性结果６例，经套式ＰＣＲ检测均为阳性，占阳性比率的１６．２％（６／３７），说明培养法只能用于初筛。     

两步PCR法检测细胞培养物中支原体污染的初步结果

本文报道用两步ＰＣＲ法快速检测细胞培养物中污染的支原体（Ｍｙｃｏｐｌａｓｍａ）。两套引物选自１６ＳｒＲＮＡ和２３ＳｒＲＮＡ保守区域，外部引物ＰＦ１，ＰＲ１扩增的ＤＮＡ片段在３６０～５００ｂｐ之间，内部引物ＰＦ２，ＰＲ２扩增的ＤＮＡ片段在１４０～２２０ｂｐ之间。试验表明，两步ＰＣＲ法能检出污染细胞培养物七种常见的支原体。１７份待检细胞培养物，两步ＰＣＲ法检测，９份（５２．９％）为阳性，直接培养法检测，５份（２９．４％）为阳性。采用快速热循仪，使ＰＣＲ反应时间大大缩短，３０次循环仅需２０分钟。反应液的用量也仅１０μｌ，提高了检测效率，节省了材料，降低了试验用费。本文就两步ＰＣＲ的灵敏度等特性也作了初步测试。     

胎儿丢失、输卵管妊娠、盆腔炎性疾病者NUC感染研究

为了解无锡地区胎儿丢失，输卵管妊娠，盆腔炎性疾病者ＮＵＣ感染状况，我们应用ＮＧ－ＰＣＲ法，ＵＵ－ｎＰＣＲ和ＣＴ－ｎＰＣＲ法对以上三种疾病者以及对照组的宫颈脱落ＮＵＣ特异性ＤＮＡ进行了检测。结果发现：胎儿丢失，输卵管妊娠，盆腔炎性疾病者ＮＧ，ＵＵ，ＣＴ均有着较高的阳性检出率，相对危险性分析提示本文研究的三种疾病与ＮＵＣ感染相关！并发现胎儿丢失者以高ＮＵＣ总阳性率（５７．０％）和低合并感染构成比（９．     

PCR检测支原体和沙眼衣原体在产科中的应用

采用常规聚合酶链反应（ＰＣＲ） 和套式ＰＣＲ（ＮＰＣＲ） 技术检测妊娠２１ ～３１ 周１９０ 份孕妇子宫颈分泌物中人型支原体（ＭＨ） 、解脲支原体（ＵＵ）和沙眼衣原体（ＣＴ）ＤＮＡ， 结果表明各感染原ＤＮＡ总阳性率为３７－９％ （７２／１９０） ； 其中ＭＨ、ＵＵ与ＣＴ ＤＮＡ检出率各为６－３％ （１２／１９０）， ２５－３ ％ （４８／１９０ ）与６－３％ （１２／１９０） ； 在ＤＮＡ阳性组中， 支原体与衣原体阳性率分别为８３－３％ （６０／７２） 与１６－７％ （１２／７２ ）。结果提示， 对上述感染者给予及时足量与足程的抗支原体与抗衣原体治疗， 将有利于胎儿的健康生长与发育。     

TT病毒的检测及部分核苷酸序列比较

目的 为阐明ＴＴ病毒（ＴＴＶ）流行病学特征和基因级异质性以及血清学诊断试剂的研制提供资料。方法 从健康查体者的血清中提取ＤＮＡ，设计一套引物，采用套式聚合酶链反应（ｎｅｓｔｅｌ－ＰＣＲ）检测ＴＴＶ，对其中７例阳性标本进行了克隆测序，并与２株ＴＴＶ的核苷酸序列作了比较。结果 １１１例正常人中ＴＴＶ的感染率为１２．６％。７株中国株ＴＴＶ间的核苷酸同源性为９０．６％～９５．４％，与日本株ＴＸ０１１（Ｏ）     

多聚酶链反应和寡核苷酸探针检测衣原体

本研究所用引物和探针序列选自衣原体１６Ｓ ｒＲＮＡ基因。以生物素末端标记制备探针。多聚酶链反应（ＰＣＲ）扩增物作琼脂糖凝胶电泳和ＤＮＡ斑点杂交，证实引物为衣原体属特异性。ＰＣＲ检测灵敏度达到相当于１个拷贝的衣原体ＤＮＡ分子。生物素化寡核苷酸探针ＤＮＡ斑点杂交的直接检测灵敏度为１００ｐｇ，结合ＰＣＲ扩增的检测灵敏度为１ｆｇ。本法高度敏感、特异，且方法简便，对于衣原体感染诊断和分子流行病学调查具有较好     

套式（Nested）PCR检测肺炎支原体与肺炎衣原体研究

本文报告根据肺炎支原体（Mp）和肺炎衣原体（Cpn）的16SrRNA基因序列，建立了套式（Nested）PCR检测方法。灵敏度试验表明，Mp套式PCR和Cpn套式PCR灵敏度分别高出Mp普通PCR和Cpn普通PCR二个数量极。     

淋病奈瑟菌感染的套式PCR法基因诊断研究

本文根据已发表的淋病奈瑟菌之隐匿性质粒ｃｐｐＢ基因序列，建立了淋病奈瑟菌感染的套式ＰＣＲ基因诊断法。本法灵敏度较普通ＰＣＲ高２－３个数量级，并且特异性更强。灵敏、特异、简便的套式ＰＣＲ法检测，在含菌量较少的男性慢性淋病患者和无症状性的女性淋病患者的诊断中可能有着较高的应用价值。     

新生儿疾病沙眼衣原体和解脲脲原体感染的PCR检测研究

为探讨沙眼衣原体（CT）和解脲脲原体（UU）与新生儿疾病的关系，我们采用套式聚合酶链式反应（PCR）技术，从100例住院新生儿的结膜拭子中共检出阳性者58例，阳性率58％（其中CT36倒、UU22例）：剖宫产7例（CT4例，UU3例）；经产道分娩51例（CT32例、UU19例）。结果表明：新生儿是CT、UU易感者．可引起新生儿肺炎、宫内感染、早产、低体重、结膜炎、肠炎。采用先进的套式PCR技术检测具有灵敏、特异、简便的优点。     

Rapid multiplex nested PCR for detection of respiratory viruses

Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken.     

A real-time PCR assay for measuring alcelaphine herpesvirus-1 DNA

Alcelaphine herpesvirus-1 (AlHV-1) is a rhadinovirus that causes malignant catarrhal fever in certain ruminant species and is an important pathogen in Africa and other areas where carrier species and clinically susceptible ruminants intermingle. In this study, a real-time PCR for AlHV-1 DNA was developed and compared to an established nested PCR. The nested PCR amplifies a region of the AlHV-1 gene coding for a transactivator protein (ORF 50), while the real-time PCR assay targets the AlHV-1 gene coding for a tegument protein (ORF 3). The real-time PCR assay reproducibly detected 10 copies of target DNA. In a dilution series of the target DNA there was linearity of the assay across 8 orders of magnitude (10(1)-10(9) copies). The nested PCR was more sensitive (approximately with 1 log) than the real-time PCR. The assay specifically amplified samples containing only AlHV-1, but not other common herpesviruses of cattle. In conclusion, we have developed a rapid, relatively sensitive, and reliable real-time PCR assay specific for AlHV-1. Similar to the real-time PCR for Ovine herpesvirus-2, this assay should prove useful for differential diagnostics of clinical MCF and for research to better define the epidemiology of AlHV-1 in wildebeest as well as in animals with wildebeest-associated MCF.     

A rapid and highly reliable field-based LAMP assay of canine parvovirus

Loop-mediated isothermal amplification (LAMP) is known as a rapid and reliable alternative to conventional single-step or nested PCR for detection of genomic DNA of various pathogens in clinical samples. In this study, LAMP assay was developed for canine parvovirus (CPV) and compared with single-step and nested PCR assays. Out of 50 fecal samples from dogs clinically suspected for CPV infections, 19 were found positive by single-step PCR, 22 by nested PCR and 26 by LAMP. LAMP products were subjected to restriction analysis and sequencing to check their specificity. LAMP assay turned out to be a rapid and fairly reproducible method, did not amplify other common canine pathogens and was more sensitive than nested PCR assay. Therefore, it can be regarded as a highly reliable method for routine field diagnosis of CPV infection. Keywords: canine parvovirus; nested polymerase chain reaction; loop-mediated isothermal amplification; sensitivity; specificity.     

PCR detection of DNA specific for Aspergillus species in serum of patients with invasive aspergillosis.

We investigated the possible presence of DNA specific for Aspergillus species in serum samples of patients with invasive aspergillosis (IA) by the nested PCR method. Fourteen strains of fungi including 5 strains of Aspergillus species and 10 strains of common bacteria were used for examination of specificity and sensitivity of the nested PCR. Two sets of oligonucleotide primers were derived from the sequence of the variable regions V7 to V9 of the 18S rRNA genes of Aspergillus fumigatus. The specific fragment was amplified from five strains of Aspergillus species in the single and nested PCR but not from other microorganisms. Target DNA was detected by the nested PCR with as little as 50 fg of the extracted DNA of A. fumigatus. We investigated the detection of DNA specific for Aspergillus species in serum samples of a murine model of aspergillosis and 20 patients with IA. The specific fragment was detected by the nested PCR in 71% of serum samples of infected mice and 70% of serum samples of patients with IA, while galactomannan antigen was detected in 43 and 60% of samples, respectively. The high sensitivity and specificity of the nested PCR indicate that the assay can provide early diagnosis with sufficient accuracy to be clinically useful for immunocompromised patients with IA.