多重聚合酶链反应和反向线性点杂交技术在常见呼吸道病毒检测中的应用

目的应用多重聚合酶链反应（mPCR）扩增基因技术和反向线性点杂交技术（RLB）相结合的方法从基因水平建立常见呼吸道病毒的检测方法。方法根据常见的7种呼吸道病毒包括流感病毒A,副流感病毒1、2、3型,腺病毒1型,呼吸道合胞病毒A、B型的特点设计特异性的引物和探针,应用mPCR/RLB技术对常见的7种病毒株进行检测,建立研究方法。结果使用特异性引物对7种病毒株的基因进行mPCR,均得到相应的目标扩增产物,扩增条带清晰,与目标片段一致。7种病毒株的PCR产物与特异性探针杂交,均可得到清晰的杂交模式,并且彼此之间没有交叉反应,与作为阳性对照的细菌标准菌株亦没有交叉反应。结论 mPCR和RLB相结合是从基因水平检测常见呼吸道病毒的方法 。     

RT-PCR技术在流行性出血热病毒分型诊断中的应用

目的　运用逆转录聚合酶链反应 (RT- PCR)技术对流行性出血热 (EHF)病人标本进行研究 ,以建立一项有效的 EHF病毒分型诊断方法。方法　收集发病 10 d以内的早期 EHF病人血浆、外周血单个核细胞 (PBMC)、尿沉淀细胞。采用异硫氰酸胍 -酚 -氯仿一步抽提各种标本的总RNA。两对特异性引物分别互补于汉滩病毒 ( 型 )及汉城病毒 ( 型 ) M基因片段的 G1区。分别在两对引物的正链引导下反转录合成特异性 c DNA,用相应的引物对进行 PCR扩增。产物经琼脂电泳 ,标准分子量对照鉴定。同时以 Vero- E6细胞及鼠脑传代的标准病毒株进行阳性对照 ,以正常人血浆作阴性对照。结果　 19例病人的 30份标本均能被 I型或 型引物扩增出特异性产物 ,其中I型阳性 13例 , 型阳性 4例 ,I、 型同时阳性 2例 ,同一病人的不同标本结果一致。结论　标准病毒株 RT- PCR分型结果与其本身的血清型一致     

乙型肝炎病毒共价闭合环状DNA套式-实时荧光定量聚合酶链反应的建立

目的 建立检测HBV共价闭合环状DNA(cccDNA)的套式一实时荧光定量PCR法.方法 根据HBV cccDNA与松环DNA(rcDNA)结构上的差异,设计2对跨缺口的特异引物及1条位于负链缺口下游的特异TaqMan荧光探针.根据Plasmid-SafeTM ATP-Dependent Dnasc(PSAD)对rcDNA与cccDNA作用的不同,对模板DNA进行酶切纯化,降解reDNA,再进行套式PCR扩增,先用外引物和模板进行第一轮常规PCR,再用内引物、荧光探针和第一轮PCR产物进行实时荧光定量PCR,根据阳性参照标准品,得出待检标本定量值.结果 检测阳性参照标准品.得出该方法灵敏度可达2 lg拷贝/mL.用上述方法检测34份乙型肝炎患者血清HBV DNA阳性标本,25份血清HBVcccDNA阳性,28份外周血单个核细胞HBV cccDNA阳性.27份健康对照者血清HBV DNA阴性标本,6份HBV cccDNA阳性.对5份HBV cccDNA阳性标本扩增产物进行克隆测序,无碱基缺失、突变.与HBV不同基因型序列(A～G)比较,同源性为90.6%～99.1%,其中,与B、C基因型同源性为95.3%～99.1%,验证了方法的特异度.结论 套式-实时定量PCR法可检测乙型肝炎患者血清、PBMC中的HBV CCCDNA,且具有敏感、特异性.     

逆转录-套式PCR鉴定福建省登革Ⅰ型病毒

目的检测急性感染者血清中的登革病毒,并判定其型别。方法从血清中提取病毒RNA,使用通用引物和型特异性引物,用逆转录-套式PCR方法扩增登革病毒特异性核酸片段,电泳后观察判定型别。同时还将扩增产物进行测序分析。结果用通用引物扩增5份标本,均出现511bp扩增带,在型特异性引物的扩增下均出现482bp的扩增带,初步判断为DVⅠ病毒。经测序分析进一步确定为DVⅠ病毒。结论运用此方法证实2004年福建省登革热流行系DVⅠ病毒引起。     

甲型流行性感冒病毒抗原酶联免疫吸附检测试剂盒的研制及应用

目的 研制检测靶位针对甲型流行性感冒(流感)病毒核蛋白的甲型流感病毒抗原检测试剂盒,建立能检测甲型流感病毒所有亚型的方法.方法 采用双抗体夹心法检测标本中的甲型流感病毒,建立甲型流感病毒抗原ELISA方法,并对该试剂盒的灵敏性、特异性、精确性、稳定性进行测试和临床考核.结果 甲型流感病毒抗原ELISA试剂盒核蛋白检测下限为7.63 ng/Ml,灵敏度是血球凝集方法的256倍,是美国Quickdel公司胶体金产品的16倍;与乙型流感病毒、呼吸道合胞病毒、呼吸道腺病毒、副流感病毒Ⅰ/Ⅲ型、肺炎支原体、鸡新城疫病毒、鸡法氏囊病毒、鸡传染性支气管炎病毒均无交叉,特异度为100%;批内、批间变异系数(CV)值均<15%,符合国家标准;在4℃以下的稳定性可达1年,能通过37℃7 d加速试验.可检测亚型分别为H1N1、H3N2、H5N1和H9N2的甲型流感病毒.人流感病毒临床试验表明,与常规细胞培养方法比较,阳性符合率为93.44%,阴性符合率为99.31%;禽流感病毒临床试验表明,与常规细胞培养方法比较,阳性符合率为95.45%,阴性符合率为98.09%.结论 甲型流感病毒抗原ELISA试剂盒灵敏性、特异性强,可用于人甲型流感病毒和禽流感病毒感染的流行病学调查.     

快速检测细菌并革兰分型的半巢式聚合酶链反应及其初步应用

目的以半巢式聚合酶链反应(PCR)检测细菌及作革兰阴、阳性分型,并与细菌培养法比较。方法以细菌16SrRNA基因为靶序列,采用一对通用引物(pm1,pm3)和一条革兰阴性型特异性引物(pm2),以半巢式PCR方法扩增实验室保留菌株的DNA并作出革兰染色分型;以人类外周血白细胞基因组DNA、HBVDNA阳性血清以及白假丝酵母菌为对照,检测此方法的特异性;采用倍比稀释菌液作敏感性实验;与细菌培养法比较,验证此方法检测临床标本的敏感性。结果对17个实验室保留菌株进行检测,以通用引物对作第1次PCR均得到371bp长度的DNA片段;再以革兰阴性菌特异引物对(pm2,pm3)作第2次PCR,9种阴性菌均得到353bp的DNA片段,而8种阳性菌未被扩增。特异性实验表明,此通用引物与人类基因组DNA、真菌及病毒无交叉反应。敏感性实验表明,采用半巢式PCR可检测出3个CFU的细菌。对120份临床标本检测,半巢式PCR检测阳性率(29.2%)显著高于细菌培养法检测阳性率(17.5%)。结论此半巢式PCR检测细菌方法,具有特异、敏感、快速的特点,并能对细菌进行革兰阴性、阳性分型,可用于临床感染性疾病的初步诊断。     

16S rRNA基因芯片诊断新生儿败血症

目的建立16SrRNA基因加基因芯片检测新生儿败血症的诊断技术，以提高临床检测细菌的速度及准确性。方法对125例拟诊为败血症的新生儿血标本及部分脑脊液标本进行细菌16SrRNA基因及基因芯片检测．包括DNA提取、设计引物和探针、聚合酶链反应（PCR）扩增、基因芯片的制备、杂交、激光扫描与读片。结果125例中PCR检测血标本阳性64例，占51．2％；血培养阳性32例，占25．6％；非特异性指标41例，占32．8％，差异有统计学意义（P〈0．01）。若以血培养阳性和／或非特异指标至少两项阳性为诊断标准，PCR灵敏度为90．3％（56／62），特异度为87．3％（55／63），正确诊断指数为0．776。3例脑脊液标本中PCR阳性2例，培养阳性仅1例。对64例PCR阳性血标本进一步作基因芯片检测，结果通用探针均阳性。其中G^＋探针阳性60份。G探针阳性4份。30份PCR和血培养均阳性的标本中其探针菌株与血培养细菌阳性结果相符。2例脑脊液标本PCR阳性，基因芯片检测结果与培养结果相符。结论168 rRNA基因PCR加基因芯片杂交可为新生儿败血症提供早期、敏感的病原学诊断依据。     

腺病毒7型合并甲型H1N1流感病毒重症社区获得性肺炎一例

报道1例通过呼吸道标本宏基因组二代测序检测出人类腺病毒7型阳性,且血清腺病毒IgA抗体阳性,同时应用咽拭子PCR法检测甲型H1N1流感病毒核酸阳性,确诊为重症社区获得性肺炎(腺病毒7型合并甲型H1N1病毒)患者的诊治过程,结合文献复习总结该病的临床特点及诊治方法,以提高临床医生对该病的认识,改善患者的预后.     

rDNA探针杂交检测病人痰标本中结核杆菌的研究

目的 应用结核分枝杆菌特异的rDNA探针杂交检测痰标本中的结核杆菌rDNA,评价其在临床标本检测中的应用价值。方法 用引物b对结核分枝杆菌16S-23SrDNA间隔区序列进行扩增,同时加入生物素标记,制成250bp的rDNA探针。对该探针的敏感性、特异性进行了研究,并对90份结核病人,30份非结核病人痰标本的PCR产物进行了斑点杂交检测。结果 rDNA探针检测引物bPCR扩增产物的敏感性为100pg。rDNA探针与受试24种分枝杆菌和11种非分枝杆菌引物bPCR扩增产物杂交只有结核分枝杆菌、胃分枝杆菌为阳性杂交,特异性较高。而rDNA探针对90份结核病人痰标本引物b扩增产物检测的阳性率为80.2%,高于PCR扩增产物电泳检测结果 (64%)。rDNA探针与30份非结核病人痰标本杂交结果均为阴性杂交。结论 rDNA探针与引物bPCR扩增相结合能提高结核病人痰标本检测的敏感性与特异性。     

用PCR技术直接检测致病真菌的实验研究

目的　建立和评价从血标本中直接PCR检测病原真菌的方法。方法　用红、白细胞裂解液 ,基因释放剂等直接处理血标本 ,提取微量的靶DNA ,然后用真菌通用引物及种特异性引物进行PCR扩增。结果　在几种重要致病真菌制备的人血标本中检测出靶DNA ,其敏感性达 10个孢子 /ml以下 ,且从标本处理到报告结果仅需 6h。结论　用PCR方法可简便快速特异敏感地从血标本中直接检测病原真菌 ,提示可为临床快速诊断深部真菌病打下基础。     

Laboratory Approaches to the Diagnosis of Adenovirus Infection Depending on Clinical Manifestations

Abstract Background: While commercial enzyme immunoassays (EIA) intended for the detection of adenovirus in fecal specimens are widely used, there are no rapid, convenient, and sensitive commercial tests available for the detection of adenoviruses in respiratory and conjunctival specimens. The applicability of EIA for the detection of adenovirus in stool and throat samples was investigated. One-day rapid culture assay (RCA) for the detection of adenovirus in respiratory and conjunctival specimens was developed and evaluated. Patients and Methods: Stool samples from patients with gastroenteritis were tested by adenovirus EIA and by cell culture using human embryonic lung cells (HEL) and Graham 293 cells. Blood and stool samples from two BMT patients were also tested for adenovirus by PCR for at least 6 months. Throat specimens from patients with respiratory infections and conjunctival specimens were used for the evaluation of 1-day RCA compared with conventional adenovirus isolation in Graham 293 cells. Results: A total of 3,860 stool samples were tested by EIA Ridascreen^?, 8,169 by Novitec™, and 2,218 by ProSpectT^? yielding 135 (3.5%), 308 (3.7%), and 77 (3.5%) positive results, respectively. From 305 Ridascreen^?- and 340 Novitec™-negative stool samples, adenoviruses were isolated in three (0.9%) and eight (2.4%) cases, respectively, including two patients undergoing BMT. Multiple sequential stool samples from one BMT patient were repeatedly negative by EIA, but positive by PCR and cell culture. Graham 293 cells were better suited for isolation of adenovirus than HEL. EIA proved unreliable for detecting adenovirus in throat swabs. The sensitivity and specificity of RCA in throat swabs were 90% (37/41) and 100% (64/64), respectively, and 76% (16/21) and 100% (132/132) in conjunctival specimens, respectively. Conclusions: Generally, EIA is sufficiently sensitive for the diagnosis of adenovirus-associated diarrhea. However, it may not be sensitive enough to detect adenovirus in immunocompromised patients undergoing BMT and shedding very few viral particles in stools. Thus, in such cases, a more sensitive assay, such as PCR, is recommended. Furthermore, EIA is not sufficiently sensitive for the reliable detection of adenoviruses in throat swabs. One-day RCA may be useful for the detection of adenoviruses in respiratory and conjunctival specimens.     

猪链球菌2型和9型菌株的多重PCR检测

目的建立一种快速、特异且敏感的检测方法。方法以猪链球菌2型和9型的荚膜多糖抗原编码基因簇中的cps2J和cps9H基因、猪链球菌2型的主要毒力因子编码基因epf和mrp为靶基因设计了四对引物,建立了检测猪链球菌的多重PCR反应体系。用编码cps2J和cps9H基因的引物可对猪链球菌2型和9型进行分型,用编码epf和mrp基因的引物可鉴定猪链球菌2型的主要毒力相关基因。用该多重PCR反应体系对10株猪链球菌分离株进行了鉴定,并以其它几株阴性对照菌株进行对照,检测了该反应体系的特异性。以新鲜培养的猪链球菌2型菌株进行倍比稀释后进行菌落计数,并将之作为模板,对该多重PCR反应体系检测的敏感性进行了鉴定。结果10株猪链球菌分离株中有5株是9型菌株,另有5株是2型菌株,2型菌株有2种基因型:3株cps2+mrp+epf+型,2株cps2+mrp-epf-型。同时还表明该多重PCR反应体系有高度的特异性和敏感性,当模板含量在10cfu时仍能检出目的菌株。结论该多重PCR反体系是一种检测和鉴定猪链球菌2型的快速、特异且敏感的方法。     

Taq Man荧光定量RT-PCR快速检测甲3型流感病毒

目的建立一种特异、灵敏、快速的荧光定量RTPCR方法用于检测甲3型流感病毒核酸。方法根据GenBank登录的流感毒株序列,应用生物学软件进行序列比对,在甲3型流感病毒血凝素(HA)基因的保守区设计引物和TaqMan探针、并进行筛选。对荧光RT-PCR反应条件进行优化,检测该方法的特异性和灵敏度。并对疑似流感含漱液标本进行检测。结果该方法对甲3型流感病毒的检测有高度的特异性,对甲1型、乙型、禽流感病毒H5、SARS病毒及其他呼吸道病毒均无交叉反应,检测的灵敏度达0.01TCID50,可从疑似流感患者含漱液中直接检测流感病毒核酸,从病毒核酸提取至完成检测仅需3h左右。结论本研究建立的TaqMan荧光定量RTPCR是一种快速检测甲3型流感病毒特异、敏感的新方法。     

儿科重症监护室先天性心脏病合并呼吸道感染患儿的病毒性病原学研究

目的：总结儿科重症监护室中先天性心脏病合并呼吸道感染患儿的病毒性病原谱。方法收集2010年6月至2012年6月因呼吸道感染入住本院儿科重症监护室的患儿咽拭子标本622份,其中先天性心脏病合并呼吸道感染患儿咽拭子34份。应用多重聚合酶链反应(PCR)技术对呼吸道病毒进行检测,并对照分析合并先天性心脏病患儿的病毒性病原学特点。结果①34份先天性心脏病组咽拭子标本中,呼吸道病毒检测阳性20份(58.8%),588份非先天性心脏病组咽拭子标本中,呼吸道病毒检测阳性368份(62.6%)。②先天性心脏病组中,最常见的病毒分别为人鼻病毒(human rhinovirus,HRV)8份,呼吸道合胞病毒(respiratory syncytial virus, RSV)6份,人博卡病毒(human bocavirus,HBoV)4份,腺病毒(adenovirus,ADV)2份;非先天性心脏病组中,最常见的病毒分别为 HRV 160份,RSV 104份,ADV 72份,HBoV50份;其他病毒阳性率较低。③先天性心脏病组中,混合病毒感染有2份(2/20,10.0%),非先天性心脏病组中,混合病毒感染有110份(110/368,29.9%)。结论本地区儿科重症监护室中先天性心脏病合并呼吸道感染患儿的病原体中病毒性病原体检出率高,以鼻病毒、呼吸道合胞病毒、人博卡病毒和腺病毒最常见,病毒谱和非先天性心脏病组相似。     

Comparative performance of PCR-based assay versus microscopy and culture for the direct detection of Mycobacterium tuberculosis in clinical respiratory specimens in Lebanon.

SETTING: American University of Beirut Medical Center, Lebanon. OBJECTIVE: To assess the performance of a polymerase chain reaction (PCR) using primers that flank 542 bp within IS6110 in Mycobacterium tuberculosis (TB) vs. microscopy and BACTEC culture, in the diagnosis of tuberculosis. DESIGN: A total of 82 clinical respiratory pulmonary specimens and 73 samples from BACTEC vials were tested by the three methods. RESULTS: Of 24 smear-positive culture-positive (SP-CP) and 11 smear-negative culture-positive (SN-CP) TB specimens, PCR detected 83% and 64%, respectively. Among 17 specimens yielding mycobacteria other than tuberculosis (MOTT), the PCR was positive in 33% SP-CP and 14% SN-CP specimens. Among the 73 BACTEC vials, PCR was positive in 36 of 38 (95%) yielding culture-positive TB, and in one of 20 (5%) yielding culture positive MOTT. None of the 30 smear-negative culture-negative (SN-CN) clinical specimens and 15 of the CN vials were positive by PCR. The overall sensitivity of PCR was 77% and 95% for TB detection in respiratory specimens and BACTEC vials, respectively, and the specificity was 94% in both. CONCLUSIONS: Because a substantial number of TB cases are missed, especially in SN-CP specimens, a PCR-based assay utilizing these primers cannot be used reliably, alone, in clinical laboratory diagnosis of mycobacterial respiratory infections.     

Detection of Epstein–Barr virus in leucoreduced blood products

This study examined the prevalence of three human herpesviruses (HHV), namely HHV‐4 (Epstein–Barr virus/EBV), HHV‐6b and HHV‐7 in leucoreduced blood products obtained from the Sainte‐Justine Hospital blood bank. A total of 100 specimens, including 34 red blood cell concentrates, 33 platelet bags and 33 plasma units, were collected and screened by a sensitive PCR assay using virus‐specific primers. Positive units were then retested by quantitative PCR. Of the 100 specimens, one platelet unit tested positive for EBV.     

乙脑病毒嵌套式RT-PCR检测方法的建立及初步应用

目的　建立适用于检测人用猪源性生物制品中外源性乙脑病毒(JEV)的嵌套式RT- PCR方法。方法　根据已公布的JEV序列(GenBank登录号为M5 5 5 0 6 ) ,设计合成两对引物,对JEV感染的乳鼠脑组织和培养JEV的BHK 2 1细胞抽提RNA进行RT PCR和RT nestedPCR ,将PCR产物进行了克隆测序;同时对猪细小病毒(PPV)、猴空泡病毒4 0 (SV4 0 )及正常乳鼠脑组织、正常BHK 2 1细胞、PK15细胞、BSC 1细胞提取核酸进行PCR ,并对2 10份临床样品进行了检测。结果　PCR产物经琼脂糖凝胶电泳检测,JEV感染的乳鼠脑组织和培养JEV的BHK 2 1细胞均扩增出10 15bp和6 2 2bp目的基因片段,而PPV、SV4 0及未感染JEV的乳鼠脑组织、正常BHK 2 1细胞、PK15细胞、BSC 1细胞均未见特异性扩增条带,2 10份猪组织样品中未检出阳性样品。RT nestedPCR检测的最低限度为10PFU病毒。从样品核酸的提取到PCR扩增及检测结果的报告可在8小时内完成。结论　本研究建立的RT NestedPCR方法具有快速、特异、敏感、可靠的特点,可用于人用猪源性生物制品中外源性JEV的污染检测。     

Acute and latent adenovirus in COPD

INTRODUCTION: The COPD airway is infiltrated with CD8+ T cells, which has led to a virus being implicated in its pathogenesis. Some investigators have suggested a role for the persistence of the adenovirus E1A in bronchial epithelial cells. We examined respiratory tract specimens from COPD patients for the presence of E1A DNA and mRNA using real-time PCR. METHODS: Nucleic acid extraction was performed on sputum specimens from patients with COPD. Copy numbers for GAPDH, and adenovirus 5 E1A DNA and mRNA were determined using a quantitative real-time PCR assay. All samples were screened for the adenovirus hexon gene using nested PCR. RESULTS: One hundred and seventy-one patients, 80 male, aged 68.9+/-9.8 years with COPD were recruited. One hundred and thirty-six were seen during an exacerbation when admitted to hospital, 33 of whom were reviewed when clinically stable along with an additional 35 stable COPD patients. Ten patients in the exacerbation group were positive for the adenovirus hexon gene (7%), as were four in the stable group (6%). Only two patients in the exacerbation group were positive for adenovirus 5 E1A. Only one patient in the stable COPD group had detectable E1A DNA/mRNA and also tested positive for the adenovirus hexon gene. CONCLUSION: Adenovirus is detected in similar frequencies in exacerbated and stable COPD patients. Adenovirus E1A DNA is infrequently detected in respiratory secretions from patients with COPD. Our data suggest that the persistence of adenovirus 5 E1A in lung cells of sputum samples in patients with COPD occurs infrequently.     

初夏一起腺病毒感染暴发性流行

2000年5月中旬,某学校在2—3天内,相继有120多人出现高烧、周身酸痛,部分病人腹泻、扁桃腺肿大,但是血象不高,少有咳嗽,流涕等呼吸道症状,疑似病毒性感染,遂进行现场调查,采集部分病人和部分未发病者的静脉血、咽拭子和肛拭子送实验室检验。从咽拭子获得有脱落上皮细胞,用免疫荧光试