Detection of Human Parvovirus B19 Nonstrutural Protein DNA by Nested-Polymerase Chain Reaction in Gravida Serum and Pregnant Tissues

A new nested-polymerase chain reaction （nested-PCR） assay was developed to detect human parvovirus B19 DNA corresponding to the nonstructural protein in clinical specimens in a routine diagnostic laboratory. The sensitivity of this highly specific assay was up to 0. 005 fg of B19 DNA. Parvovirus B19 was identified in sera of 20 pregnant women with abnormal pregnant outcome. Among these 20 cases, intrauterine parvovirus infection did exist in 7 pregnant women because parvovirus B19 DNA was detected in the pregnant tissues of them such as placenta tissues, chorionic villi, amniotic fluid, fetal spleen, liver and abdominal fluids.

Detection and amplification of Helicobacter pylori urease gene A in gastric biopsies by using nested polymerase (?)hain reaction

A nested polymerase chain reaction(N-PCR)for the spegific detection of Helicobacterpylori(H.pylori)was developed with two primer pairs(nested primers)derived from ureasegene A of H.pylori.The N-PCR was used to detect 21 different samples of H.pylori including20 clinical isolates and 1 reference strain NCTC 14126,but it was negative for other bacterialspecies,showing the N-PCR assay to be 100% specific.Tenfold serial dilution experiments re-vealed the detection of as little as 0.1 fg of H.pylori DNA by N-PCR.To evaluate the PCR as-say for clinical samples,gastric biopsies were tested with N-PCR,and the results were comparedwith those of culture,urease test and histologic examination(reference standard,RS).In 30biopsy specimens,H.pylori DNA sequences were detected by PCR in all of 20(100%)positivetissue and none of the 10 negative tissues.PCR is a specific and sensitive method that can detectthe presence of H.pylori without the need for culture and would have significant importance di-agnostically and epidemiologically.

Development and Clinical Application of a Single-tube Nested PCR Method to Amplify the DNA Polymerase Ⅰ Gene of Treponema Pallidum

Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.

进口锦鲤暴发病病原的nested-PCR鉴定

为了查明锦鲤暴发性疾病的病因 ,将患病锦鲤的脑、肝、脾和肾等组织悬液接种到CO、CK和EPC等鱼类细胞系中培养 ,均未发现细胞病变 ;从病灶中取样进行病原菌的分离和培养 ,证明锦鲤有副溶血弧菌和嗜水气单胞菌的感染。制备锦鲤疱疹病毒 (KHV)的 2对引物KHV9/ 5F和KHV9/ 5R ,KHV1和KHV2 ,用嵌套式聚合酶链式反应 (nested -PCR)在脑和脾脏组织的抽提物中扩增出长度为 4 12bp的特异性的DNA片段。将该片段的nested -PCR扩增产物纯化后 ,克隆、测序。用NCBI -Blast软件将测序结果在Genebank中进行搜寻、比较 ,结果发现与注册号为AF4 1180 3的KHV基因序列的一部分片段有 99%的同源性。因此初步判断这次锦鲤暴发性疾病是由KHV引起的 。

Nested PCR检测微量隐孢子虫卵囊

根据隐孢子虫 18S r DNA序列 ,设计出隐孢子虫属和鼠隐孢子虫种的特异性引物 ,进行 PCR和 Nested PCR反应 ,先后分别扩增出 1条 5 4 0 bp和 1条 2 5 0 bp的条带。研究表明 ,Nested PCR具有高度的特异性和敏感性。应用Nested PCR可检测 1～ 10个鼠隐孢子虫卵囊 ,其敏感性是饱和蔗糖漂浮法的 10 5倍以上。

Nested polymerase chain reaction in detection of Plasmodium vivax sporozoites in mosquitoes

Objective To detect malaria DNA in mosquitoes.Methods A nested polymerase chain reaction (nested PCR) procedure which amplifies a 121 bp DNA of a SSUrRNA gene specific to Plasmodium vivax was used.Results In laboratory-infected mosquitoes, nested PCR could detect as few as 3 sporozoites or 1 infected mosquito mixed in a group of 99 normal ones. Furthermore, no specific 121?bp band was seen with DNA templates from other malaria parasites or negative mosquitoes.Conclusion Sensitivity and specificity obtained indicated an advantage of the nested PCR over DNA probes or direct PCR for the detection of Plasmodium vivax sporozoites in mosquitoes with low-grade parasitic infections.

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）。

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.

Establishment of a nested-ASP-PCR method to determine the clarithromycin resistance of Helicobacter pylori

AIM: To investigate clarithromycin resistance positions 2142, 2143 and 2144 of the 23 Sr RNA gene in Helicobacter pylori(H. pylori) by nested-allele specific primer-polymerase chain reaction(nested-ASP-PCR).METHODS: The gastric tissue and saliva samples from 99 patients with positive results of the rapid urease test(RUT) were collected. The nested-ASP-PCR method was carried out with the external primers and inner allele-specific primers corresponding to the reference strain and clinical strains. Thirty gastric tissue and saliva samples were tested to determine the sensitivity of nested-ASP-PCR and ASP-PCR methods. Then, clarithromycin resistance was detected for 99 clinical samples by using different methods, including nestedASP-PCR, bacterial culture and disk diffusion. RESULTS: The nested-ASP-PCR method was successfully established to test the resistance mutation points 2142, 2143 and 2144 of the 23 SrR NA gene of H. pylori. Among 30 samples of gastric tissue and saliva, the H. pylori detection rate of nested-ASP-PCR was 90% and 83.33%, while the detection rate of ASP-PCR was just 63% and 56.67%. Especially in the saliva samples, nested-ASP-PCR showed much higher sensitivity in H. pylori detection and resistance mutation rates thanASP-PCR. In the 99 RUT-positive gastric tissue and saliva samples, the H. pylori-positive detection rate by nested-ASP-PCR was 87(87.88%) and 67(67.68%), in which there were 30 wild-type and 57 mutated strains in gastric tissue and 22 wild-type and 45 mutated strains in saliva. Genotype analysis showed that three-points mixed mutations were quite common, but different resistant strains were present in gastric mucosa and saliva. Compared to the high sensitivity shown by nested-ASP-PCR, the positive detection of bacterial culture with gastric tissue samples was 50 cases, in which only 26 drug-resistant strains were found through analyzing minimum inhibitory zone of clarithromycin. CONCLUSION: The nested-ASP-PCR assay showed higher detection sensitivity than ASP-PCR and drug sensitivity testing, which could be performed to evaluate clarithromycin resistance of H. pylori.

肺炎衣原体的套式(Nested)PCR检测及其临床意义

肺炎衣原体（C_(pn)）可致上、下呼吸道的炎症，并且是非典型性肺炎综合征（APS）的主要病原体之一。C_(pn)已占肺炎病原体的第三或第四位。近年的深入研究表明，C_(pn)还能引起呼吸道之外的脏器炎症。由于C_(pn)为专性细胞内寄生菌，因此培养困难。目前国内尚缺乏C_(pn)感染诊断方法。本文介绍一种灵敏、特异、简便的C_(pn)套式PCR检测方法。应用本法检测55例呼吸道感染者之咽拭子，共检出9例阳性（阳性率16．2％）。表明C_(pn)的呼吸道感染在我国并不少见。C_(pn)套式PCR方法的建立，对于我国的C_(pn)人群感染率和C_(pn)感染的分子流行学研究有着极其重要的意义。

套式聚合酶链式反应(Nested PCR)检测肺炎衣原体

肺炎衣原体（Chlamydiapneumoniae．CPn）可引起人类急性呼吸道炎症，临床表现以肺炎为主，已在世界上许多国家和地区引起流行，本文介绍通过套式PCR检测CPn，共检测109例呼吸道感染者之咽拭标本，检出13例阳性（阳性率12％）．提示CPn的呼吸道感染在我国并不少见。实验结果表明，套式PCR用于CPn检测方法的建立，有敏感、特异、简便、快速的特点，具有良好的应用前景。

纳米免疫磁珠联合RT-nest-PCR法检测胰腺癌外周血微转移的敏感度及特异性

目的:通过胰腺癌外周血微转移模型,探讨纳米免疫磁珠联合RT-nest-PCR检测微转移的敏感度及特异性。方法:将胰腺癌细胞PANC-1稀释分别加入到正常人外周血单核细胞中,再按比例加入Fcλ非特异性受体阻断剂以及上皮细胞EP-CAM抗体,4℃下混匀孵育30 min,经分选柱阳性分选后提取RNA,RT-nest-PCR法检测肿瘤基因人端粒酶亚单位(h-TERT)和癌胚抗原(CEA)。结果:联合纳米免疫磁珠和RT-nest-PCR,平均1×107外周血单核细胞可以检测到1个肿瘤细胞。10例良性疾病对照组均未见表达。结论:免疫磁珠联合RT-nest-PCR是敏感度及特异性都较高的检测微转移的方法,可显著提高肿瘤微转移的早期检测率。

巢式多重聚合酶链反应快速诊断儿童脑膜炎/脑炎

目的探讨巢式多重聚合酶链反应(PCR)在儿童脑膜炎/脑炎诊断中的价值。方法采用回顾性病例对照研究。收集2017年5月—2020年5月上海市儿童医院82例确诊或疑似中枢神经系统感染患儿的脑脊液样本。收集患儿临床资料,了解患儿抗菌药物使用情况。采用巢式多重PCR检测病原体,并同步进行细菌培养、脑脊液常规和生化检测。结果82例脑脊液样本中,有31例检出病原体,其中20例检出病毒、11例检出细菌。细菌组脑脊液样本白细胞计数、蛋白、乳酸脱氢酶和患者抗菌药物使用天数、住院天数均显著高于病毒组(P<0.05);葡萄糖水平低于病毒组(P<0.05);阿昔洛韦使用例数少于病毒组(P<0.05)。巢式多重PCR与培养法判断细菌性脑膜炎/脑炎的一致性较好(Kappa=0.659,P<0.001),与实验室指标判断病毒性脑膜炎/脑炎的一致性亦较好(Kappa=0.737,P<0.001)。结论巢式多重PCR可以作为辅助诊断中枢神经系统感染患儿脑脊液样本病原体感染的新方法,可提高儿童脑膜炎/脑炎的诊断效率。

1240例女性生殖道支原体感染分析

目的 为了解生殖道支原体在晚期孕妇、育龄妇女、念珠菌性阴道炎和淋球菌性阴道炎患者中的感染状况。方法 采用套式聚合酶链反应技术 ,对 96 2例晚期孕妇、85例育龄妇女、90例念珠菌性阴道炎和 10 3例淋球菌性阴道炎患者的宫颈拭子标本进行了生殖道支原体检测。结果 在晚期孕妇、育龄妇女、念珠菌性阴道炎和淋球菌性阴道炎患者中生殖道支原体的检出率分别为 4 .16 %、3.5 2 %、11.11%和 13.5 9%。结论 生殖道支原体在晚期孕妇、育龄妇女、念珠菌性阴道炎和淋球菌性阴道炎妇女中均有一定的检出率。

3种肺炎支原体感染检测方法的临床效果比较

目的比较实时荧光定量聚合酶链反应(FQ-PCR)法、培养-增强套式PCR(培养-增强nPCR)法和培养法检测肺炎支原体感染的临床应用价值。方法分别采用实时FQ-PCR法、培养-增强nPCR法和培养法对临床诊断为肺炎支原体肺炎和疑似肺炎支原体肺炎的97例患儿的咽拭子标本进行检测。结果实时FQ-PCR法检测的阳性率明显高于培养法(P<0.001),实时FQ-PCR法的检出率和培养-增强nPCR法比较无显著差别(P>0.05),但实时FQ-PCR法更加简便、快速。结论实时FQ-PCR法较培养-增强nPCR法和培养法更适用于肺炎支原体的早期快速诊断。

中国北方5城市慢性乙型肝炎患者的基因分型

目的 了解中国慢性乙型肝炎 (CHB)患者中的基因分型情况。方法 根据GenBank中 18株HBVS基因核苷酸序列 ,设计共同的外引物及其因型特异性内引物 ,对中国北方 5个城市 5 95例CHB患者血清进行基因分型 ,并对A、B、C及B、C混合型各一株进行测序。结果 5 95例血清标本中 ,A型 8例 ,占 1 4 % ;B型 5 3例 ,占 8 9% ;C型 36 0例 ,占 6 0 5 % ;B、混合型 112例 ,占 18 8% ;A、C混合型 14例 ,占 2 4 % ;A、B混合型 15例 ,占 2 5 % ;A、B、C混合型 6例 ,占 1 0 % ;未分型者 2 7例 ,占 4 5 %。 5城市间在B、C及B、C混合型的分布上有显著性差异 (P <0 0 5 ) ,4份测序结果与PCR基因分型法一致。结论 在 5个城市CHB患者中HBV基因型以C型为主 ,B型次之 ,并见各种混合基因型感染 ,各地区在主要基因型分布上存在显著性差异。

甲氨蝶呤对银屑病患者皮损内VEGF mRNA影响的研究

目的研究甲氨蝶呤(methotrexate,MTX)对银屑病皮损内血管内皮生长因子(VEGF)表达的影响,探讨其对银屑病的治疗机理。方法随机选择12例正常对照者和13例中度、重度银屑病患者,所有患者治疗前和治疗6周后行皮肤活检。利用巢式聚合酶链反应法测定12例正常对照和13例银屑病患者治疗前及治疗6周后皮损内VEGFmRNA的定量。结果银屑病皮损内VEGFmRNA高于正常对照者皮肤内的水平(P=0.005)。银屑病患者经过MTX治疗6周后,皮损明显改善,皮损内VEGFmRNA的水平低于银屑病患者治疗前皮损内的水平(P=0.030),但与正常对照者皮肤内的水平无统计学差异(P=0.687)。结论MTX能抑制银屑病皮损内VEGFmRNA的过度表达,这可能是MTX治疗银屑病的作用机制之一。

河南省3例输入性三日疟的诊治分析

采用吉氏染色镜检、CareStartTM疟疾快速诊断试剂盒和巢式PCR等3种方法,对2011年河南省3例自安哥拉和赤道几内亚归国的三日疟患者血样进行检测。2例自安哥拉归来的患者分别于回国后15 d和27 d发病;另1例患者曾在赤道几内亚发病,归国2个月后再次发病。3例患者均有发热、头痛和畏寒等症状,但发热规律不典型。2例患者出现总胆红素升高、脾大等临床表现。3例患者镜检均可见典型的三日疟原虫形态,巢式PCR检测均扩增出与三日疟预期一致的特异性条带,但CareStartTM疟疾快速诊断试剂盒检测结果均为阴性,3例患者均经复方青蒿素类药物(ACT)治愈。

转基因玉米59122品系的特异性检测

使用反向聚合酶链式反应(PCR)技术克隆了转基因玉米59122的外源基因与玉米基因组之间的两段侧翼序列,并据其左侧侧翼序列设计了具品系特异性的引物,运用半巢式PCR技术建立了59122的品系特异性二重PCR检测方法,扩增片段100bp,横跨pat终止子与转基因玉米侧翼基因之间。以转基因玉米59122、MON863、MON810、GA21、NK603,转基因大豆Roundup Ready和转基因油菜GT73等为材料,证明本方法与其他转基因作物具有高特异性。本方法在检测59122时,确定出连接体系中线性DNA的最佳质量浓度为1ng/μL左右,检出限达到0.1%,灵敏度为38个单倍体基因组拷贝数。因此可准确、快速、高效地检测转基因玉米及其产品,或作为常规PCR定性检测后的验证方法。

应用NPCR发现我国Kawasaki型恙虫病立克次体

本文报告用恙虫病立克次体表面蛋白５６ＫＤａ型特异抗原（ｔｓａ５６ＫＤａ）基因编码区的引物，采用嵌合式聚合酶链反应（ＮＰＣＲ）鉴定江苏地区的２株恙虫病立克次体。结果该２株恙虫病立克次体与Ｇｉｌｌｉａｍ，Ｋａｒｐ，Ｋａｔｏ和Ｋｕｒｏｋｉ型特异引物无任何ＤＮＡ扩增带，而与日本ｋａｗａｓａｋｉ株型特异引物扩增后有５２３ｂｐ的ＤＮＡ扩增带，表明我国存在Ｋａｗａｓａｋｉ型恙虫病立克次体。