Lumiget PPD化学发光法检测HCMV DNA

在ＤＩＧ标记及检测系统中，以Ｌｕｍｉｇｅｔ ＰＰＤ作为ＡＰ底物，经ｄｏｔ－Ｂｌｏｔ杂交后进行化学发光法检测ＨＣＤＭＶ ＤＮＡ，并与ＮＢＴ义物显色法比较，结果显示：Ｌｕｍｉｇｅｔ ＰＰＤ法可检出０．１Ｐｇ，同源ＤＮＡ，非同源性ＨＢＶ ＤＮＡ，ＨＳＶ－Ⅰ，ＨＳＶ－Ⅱ，ＤＮＡ不能检出；Ｌｕｍｉｇｅｔ ＰＰＤ底物作用只需３ｈ即可获得清晰稳定的结果，优于ＮＢＴ底物显色法。     

Luteinizing hormone-releasing hormone gene expression differs in young and middle-aged females on the day of a steroid-induced LH surge

LHRH mRNA levels were examined in young and middle-aged female rats at 4 times (10:00 h, 14:00 h, 18:00 h and 20:00 h) on the day of a steroid-induced LH surge by in situ hybridization with a digoxigenin-labeled riboprobe. Young, but not middle-aged females, exhibited dynamic temporal changes in the number of LHRH mRNA positive neurons detected in the organum vasculosum of the lamina terminalis–preoptic area (OVLT–POA) continuum. Specifically, fewer LHRH mRNA positive neurons were detected at 18:00 h compared with the number detected at 14:00 h and 20:00 h (P<0.01) in the OVLT–POA of young females. All LHRH mRNA positive neurons present in 4 anatomically matched sections through the rostral POA of young and middle-aged animals were digitized for detailed computer-assisted analysis of the hybridization reaction product. The mean hybridization area (P<0.00025) and integrated optical density per cell (P<0.006) were reduced in middle-aged compared to young females consistent with a relative age-related decline in LHRH mRNA levels. Moreover, an age-related reduction in cellular and/or regional hybridization area was noted at each of the time points examined (P<0.05–P<0.001). These data confirm earlier reports of dynamic changes in LHRH mRNA levels on the day of an LH surge. Furthermore, they support a role for age-related alterations in LHRH gene expression in the disruption of regular estrous cyclicity in middle-aged females.     

改进探针标记法斑点杂交在轮状病毒VP7分型中的应用

目的建立一种更加简便的A组人轮状病毒（HRV）核酸斑点杂交VP7分型方法,以便用于对HRV流行情况进行调查。方法在HRVVP7编码基因各G基因型问高度变异而型内高度保守区域设计分型探针,在该区域的两侧相对保守区域设计一对通用引物,利用PCR分别将地高辛标记HRV5种常见型别（G1～4,G9型）的DNA探针,建立基于VP7的斑点杂交方法;选取经抗原检测和聚丙烯酰胺凝胶电泳（PAGE）检测均为HRV阳性的2006至2008年住院腹泻患儿粪便标本200份,RT—PCR扩增VP7全基因,并对扩增阳性产物应用斑点杂交方法进行G型别分析。结果建立的斑点杂交方法在5种型别探针间无交叉反应,各型探针的检测灵敏度可达到10Pg。200份PAGE阳性标本中162份RT—RCR扩增VP7基因阳性,斑点杂交显示G1型41例（25．3％）,G2型2例（1．2％）,G3型63例（38．9％）,G9型35例（21．6％）,混合感染19例（11．7％）,杂交未分出型2例（1．2％）,未检测到G4型HRV。结论本研究所建立的斑点杂交方法敏感度和特异度强,适合在HRV大规模分子流行病学调查时应用。通过该方法的初步应用,发现北京地区婴幼儿HRV除了常见的G1、G2和G3型外,还有G9型感染。     

地高辛标记探针原位杂交检测肝组织中HCV RNA

用地高辛标记ＨＣＶ５′－ＮＣ区ｃＤＮＡ探针原位杂交，检测了２４例ＨＣＶ感染的慢性肝病患者肝组织中ＨＣＶ ＲＮＡ。结果：１７例同时检测血清抗－ＨＣＶ和ＨＣＶ ＲＮＡ均阳性患者中，１４例（８２．３％）肝组织检出ＨＣＶ ＲＮＡ。７例仅有抗－ＨＣＶ阳性患者，肝组织中未检出ＨＣＶ ＲＮＡ。ＨＣＶ ＲＮＡ特异性信号主要位于肝细胞浆。感染ＨＣＶ肝细胞周围见较多的淋巴细胞和单核细胞浸润。结果提示ＨＣＶ在肝细胞浆复     

原位杂交在成年和幼年型喉乳头状瘤HPV病因学的对比研究

探讨成年和幼年型喉乳头状瘤ＨＰＶ感染发病差异及其影响因素。方法：用地高辛配基（Ｄｉｇｏｘｉｇｅｎｉｎ）标记ＨＰＶ６和ＨＰＶ１１型作探针，原位核酸杂交方法在２９例成年型喉乳头状瘤（ＡＬＰ）和２１例幼年型喉乳头状瘤（ＪＬＰ）石蜡包埋标本检测ＨＰＶ同源序列。结果：ＡＬＰＨＰＶ６和ＨＰＶ１１阳性率分别为４１４％（１２／２９）和４８３％（１４／２９）；ＪＬＰＨＰＶ６及ＨＰＶ１１阳性率均为７６２％（１６／２１）。ｘ２统计示：两型喉乳头状瘤ＨＰＶ６及ＨＰＶ１１阳性率明显不同（ＨＰＶ６ｘ２＝５９９，ＨＰＶ１１ｘ２＝３９５，Ｐ均小于００５）。结论：１）ＡＬＰ和ＪＬＰＨＰＶ感染发病存在差异。２）ＡＬＰ除了ＨＰＶ感染外，其促发因素不可忽视，ＪＬＰ更倾向于依赖ＨＰＶ感染而发病。     

Detection of Helicobacter pylori by enzyme-linked immunosorbent assay of thermophilic helicase-dependent isothermal DNA amplification

An enzyme-linked immunosorbent assay (ELISA) of thermophilic helicase-dependent isothermal DNA amplification (tHDA) was developed for detection of Helicobacter pylori. The primers targeting ureC were used for the amplification of bacterial DNA by the isothermal digoxigenin (DIG)-labeling tHDA process, resulting in the accumulation of DIG-labeled DNA amplicons. The amplicons were denatured using heat and then hybridized with a specific biotinylated DNA probe, which was noncovalently immobilized on streptavidin-coated microtiter plate. The hybrids were colorimetrically detected by the addition of an anti-DIG antibody HRP conjugate and 2,2-azino-di-(3-ethylbenzthiazolinsulfonate) substrate solution. Results obtained from the gastric biopsy samples showed 90% and 95.7% of sensitivity and specificity, respectively, in comparison with culture results, and 96.6% and 96.8% of sensitivity and specificity, respectively, in comparison with those of the histologic studies. This assay significantly reduces the time needed for the identification of H. pylori and has the potential to facilitate early detection of this gastrointestinal pathogen.     

HIV-1型前病毒基因检测芯片的研制及应用

目的研制艾滋病病毒1型(HIV-1)前病毒基因检测芯片，并将其应用到临床样本的检测中。方法合成多对引物，经筛选实验后选出6对适宜的引物用于逆转录-聚合酶链反应(RT—PCR)，扩增HIV基因组gag区(保守区)6个HIV目的基因片段，扩增小鼠GAPDH基因片段作为阳性内参片段，PCR扩增辣椒红素基因片段作为阴性对照片段。将上述片段克隆到pMD18-T载体上，从中选取3个HIV-1目的片段、阳性对照片段和阴性对照片段进行PCR扩增，扩增产物经纯化后点在尼龙膜上，制备成核酸检测芯片。地高辛PCR标记样本核酸与芯片杂交后，用酶联显色，分析结果。结果共检测了98份阳性样本和30个阴性样本，敏感性达93．9％，特异性为100．0％。结论该HIV-1前病毒基因检测芯片成本较低，具有较高的特异性和灵敏度，可以用于HIV-1感染和母婴传播的早期诊断。