Studies on Particle Components of Cacao Swollen Shoot Virus

Virions of the cacao swollen shoot virus (CSSV) strain 1A were purified and used for studies of its particle components. CSSV virions had a buoyant density of 1.365 g/cm³ in buffered CsCl. Following SDS polyacrylamide gel electrophoresis (PAGE), CSSV-specific proteins were identified in electroblot immunoassays (EBIA) with cross-absorbed polyclonal antibodies and especially well with monoclonal antibodies (MABs) to CSSV-1A. Based upon EBIA experiments with a selected MAB, CSSV virions appeared to have one capsid protein species with a relative molecular mass (M), of about 43 kd that was shown to be not glycosylated. However, this protein is sensitive to proteolytic degradation as degradation products ranging from 37 to 33 kd were found in addition to the 43 kd protein. Studies on the viral genome of CSSV revealed that CSSV virions contain a DNA of about 7. 5 kbp. Nucleic acid probes obtained by cloning parts of the viral genome yielded specific hybridization reactions with extracts and preparations from plants infected with strain 1A of CSSV but not with those from non-inoculated plants. One clone of 738 bp was sequenced and shown to contain a motif similar to the putative RNA binding domain of pararetroviruses. Based upon particle morphology and properties of the virion components, CSSV can be grouped with other nonenveloped bacilliform viruses for which the name badnaviruses has been proposed.     

聚合酶链反应技术检测禽网状内皮组织增殖病病毒

目的建立聚合酶链反应(PCR)技术检测禽网状内皮组织增殖病病毒(REV)的方法。方法提取感染REV-T和脾坏死病毒(SNV)的SPF鸡胚成纤维细胞DNA为模板,利用前病毒长末端重复序列(LTR)区引物进行扩增。采集肿瘤病鸡,以及人工感染REV 28 d后鸡肝脏、脾脏、肾脏、心脏、胸腺、法氏囊等器官,进行扩增。同时将采集的脏器组织,进行HE染色和免疫组化试验(IHC)。结果REV-T感染的组织未检测出电泳条带,而SNV感染的细胞中检测到了一条300bp特异而清晰的电泳条带,而且SNV感染的鸡组织中,PCR方法检测到了特异的条带。通过HE染色和免疫组化技术观察到了肿瘤组织,肿瘤细胞的形态、分布。结论PCR检测REV更快捷,特异更好。     

The Use of Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Cacao Swollen Shoot Virus (CSSV) in Theobroma cacao

Cacao swollen shoot virus (CSSV) was readily detected in different parts of Theobroma cacao using the ELISA technique. Different plant tissues contained varying amounts of CSSV; highest concentrations were found in leaf lamina. Methods to preserve the serological activity of CSSV were evaluated, and best results obtained with samples stored in a buffer or freeze-dried.     

Detection and Comparison of some Ghanaian Isolates of Cacao Swollen Shoot Virus (CSSV) by Enzyme-Linked Immunosorbent Assay (ELISA) and Immunoelectron Microscopy (IEM) Using an Antiserum to CSSV Strain 1A

Various isolates of Cacao Swollen Shoot Virus (CSSV) were detected without difficulty in leaves of Theobroma cacao L. by ELISA and immunosorbent electron microscopy (ISEM) using an antiserum to severe strain 1A. Many isolates were detected with relatively high values at dilutions of 1:30, whereas some other isolates were hardly or not at all detected at this dilution. Strain 1A was detected at dilutions of up to 1: 2560 of crude leaf extracts. All isolates yielding high reactions seem to be serologically closely related to strain 1A. Strains of the mottle-leaf type (AD 191, AD 196, AD 7, AD 36, AD 135, Kpeve) and others were poorly detected; their relationship to strain 1A is discussed. A close correlation was found between results obtained by ELISA and ISEM.     

用套式多聚酶链反应技术监测乙型肝炎病毒的母婴垂直传播

用套式多聚酶链反应（Ｎｅｓｔｅｄ－ＰＣＲ）技术对１６９对ＨＢｓＡｇ及ＨＢｓＡｇ／ＨＢｅＡｇ阳性孕妇及其新生儿外周血清进行了ＨＢＶ－ＤＮＡ检测。１０３对ＨＢｓＡｇ阳性孕妇及其新生儿外周血清中ＨＢＶ－ＤＮＡ阳性率分别为７２．８％和３３．０％;６６对ＨＢｓＡｇ和ＨＢｅＡｇ双阳性的孕妇及其新生儿外周血清中ＨＢＶ－ＤＮＡ阳性率分别为８６．４％和４３．９％。对５５例ＨＢｓＡｇ及ＨＢｓＡｇ／ＨＢｅＡｇ阳性产妇产后的初乳进行了ＨＢＶ－ＤＮＡ检测,结果ＨＢＶ－ＤＮＡ阳性率为３６．４％。结果表明ＨＢｓＡｇ和ＨＢｅＡｇ双阳性的孕妇及其新生儿外周血清ＨＢＶ－ＤＮＡ检出率较ＨＢｓＡｇ单阳性的孕妇及其新生儿要高,其初乳中ＨＢＶ－ＤＮＡ的检出率也高。还对１０５例注射了乙肝疫苗及高价乙肝特异性免疫球蛋白的６月龄婴儿的外周血清进行了ＨＢＶ－ＤＮＡ检测,结果有２３例阳性。     

RT—PCR技术检测呼吸道合胞病毒RNA方法的建立

为了建立一种快速诊断呼吸道合胞病毒（RSV）感染的方法,根据RSVN基因的核苷酸序列,设计合成了一对引物,经RT-PCR扩增,可检出RSVRNA该引物不能检测流感病毒、副流感病毒RNA。应用该法可检出疑为RSV感染的婴幼儿鼻咽分泌物中的病毒RNA且比病毒分离法敏感,特异性与免疫荧光法一致。结果表明RT－PCR法具有快速、敏感、特异的优点,可用于RSV感染患儿的临床诊断。     

逆转录-聚合酶链反应检测鼠肝炎病毒方法的建立

建立特异、灵敏的逆转录聚合酶链反应（RT－PCR）技术,结合碱性磷酸酶标记的探针杂交检测鼠肝炎病毒（MHV）,采用MHV－3,MHV－A59病毒株感染DBT细胞,37℃培养,待细胞出现病变时收集提取病毒RNA。依据MHV基因序列设计一对高度保守区特异性引物,进行RT－PCR扩增,结果可见147bp的鼠肝炎病毒产物特异扩增带。敏感性实验检测到10pg的鼠肝炎病毒RNA,同时用ELISA方法对照。结果提示应用RT—PCR技术结合探针杂交检测鼠肝炎病毒。具有简便、快速、灵敏等优势。本研究在国内尚未见报道。     

Use of Polymerase Chain Reaction for the Detection of Infectious Hypodermal and Hematopoietic Necrosis Virus in Penaeid Shrimp

A rapid and reliable polymerase chain reaction (PCR) method was developed for the detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in penaeid shrimp. The oligonucleotide primers amplify a 1681-bp fragment of IHHNV, which encompasses the coding sequence for one of the viral coat proteins. The PCR method detects IHHNV in hemolymph and homogenized tissue obtained from the cephalothorax or pleopods of infected shrimp. The technique was also successfully applied to tissue samples preserved in 70% ethanol. The correct size fragment was amplified using IHHNV obtained from six different geographic regions in three different species of penaeid shrimp. No DNA extraction method was necessary for this technique. The use of hemolymph or pleopods provides a nondestructive screening method by which to test juveniles and adult broodstock for the presence of IHHNV. Received September 21, 1999; accepted January 21, 2000     

PCR在猴B病毒鉴定中的应用研究

目的为鉴定新分离毒株是否为B病毒.方法根据ScinicarielloF报道的引物,用PCR方法扩增BV147、HSV-1、HSV-2,对扩增产物进行SacⅡ内切酶消化.结果这一对引物可同时对这3种病毒进行扩增,但只有BV147的扩增产物可被SacⅡ内切酶切开.对BV147扩增片段克隆测序的结果证实,其与美国B病毒E2490株部分基因(UL27)相对应位置的核苷酸同源性为100%.结论初步建立了检测B病毒DNA的PCR方法并测定了新分离病毒毒株的部分基因序列,证明新分离的病毒为B病毒.     

Detection ofMycoplasmain Avian Live Virus Vaccines by Polymerase Chain Reaction

The polymerase chain reaction (PCR) was evaluated to detect mycoplasma contamination of avian live virus vaccines. The specificity of the primers showed that 34 strains belonging to nine species of avian mycoplasma DNA could be detected. The sensitivity of PCR to detect mycoplasma DNA was 10^0·2colony forming units (cfu) ofMycoplasma synoviaeand 10^0·7cfu ofMycoplasma gallisepticum. WhenM. synoviaeandM. gallisepticumwere spiked into several avian live virus vaccines, PCR gave a positive reaction except for the avian pox and the avian encephalomyelitis vaccines which were prepared from organ homogenates. Short-term incubation of avian encephalomyelitis vaccine improved the sensitivity of PCR to detect bothM. synoviaeandM. gallisepticum. Therefore, PCR, combined with the short-term incubation, were shown to be most effective in detecting mycoplasma contamination in all of avian live virus vaccines.     

A New Primer Pair for Detection of Chlamydia pneumoniae by Polymerase Chain Reaction

In order to improve the detection and identification of Chlamydia pneumoniae, new primers for polymerase chain reaction (PCR) were designed based on the DNA base sequence within the 53-kDa protein gene, which is specific for C. pneumoniae. The primers permitted the identification of 24 C. pneumoniae strains collected from different geographical locations, but no reaction was observed with C. trachomatis, C. psittaci nor C. pecorum. The primers were unable to amplify the DNA of bacteria commonly related to respiratory tract infections. The positive amplification was achieved with only 9 EBs/assay. Therefore, the new primers seem to be useful in the diagnosis of C. pneumoniae infections.     

RT-PCR和酶切方法区分猪瘟疫苗毒与野毒的研究

建立了套式RT-PCR与限制性内切酶酶切相结合的区别猪瘟兔化弱毒疫苗株与猪瘟病毒田间分离株的诊断方法。通过对猪瘟兔化弱毒疫苗株与猪瘟石门株E2基因主要抗原编码区序列进行限制性内切酶酶切位点分析,分别找出猪瘟兔化弱毒疫苗株与猪瘟石门株各自独有的限制性内切酶酶切位点,结果二者分别有10和16个独有的限制性内切酶的酶切位点;分别对17株猪瘟病毒E2基因主要抗原编码区序列进行这26个限制性内切酶酶切位点分析,结果表明有3个限制性内切酶（HgaI、Hin8I及Hsp92I）的酶切位点在HCLV株序列是独有的;利用HgaI限制性内切酶分别对HCLV、HCVSM及5株不同基因群的猪瘟病毒田间分离株进行酶切鉴定,结果只有HCLV株能够被HgaI酶切成2个片段,而其它的毒株则不能被切开。同时测定了套式RT-PCR方法的敏感性及特异性,结果其敏感性可达到0．2MLD,而对BDV以及BVDV均不能特异扩增。本方法的建立无疑对猪瘟在我国的控制和消灭具有重要的意义。     

人传染性软疣病毒快速PCR检测方法的建立

为了能够对传染性软疣病毒(MCV)感染做出准确快速的实验室诊断,从14例临床MCV患者皮疹处提取获得病毒DNA,设计引物并进行PCR扩增获得预期的167bp的片段,经测序并与已报道的序列比对,完全一致。而使用痘病毒科其他病毒的特异引物(正痘病毒和副痘病毒属)则没有特异扩增条带。将病毒DNA进行系列稀释后行PCR扩增,结果表明PCR检测方法的敏感性为5×10-4ng/μL。     

用PCR技术检测活检组织和鼻咽上皮细胞内EB病毒基因

选用Epstin-Barr病毒(EBV)基因组内部重复序列1(IR1)片断作为多聚酶链反应(Polymerase Chain Reaction,PCR)扩增引物,用于检测了31例不同病例活检组织和4例新鲜鼻咽组织经体外培养6周以上的新生上皮细胞内EBV基因,其中检出EBVDNA:高分化鼻咽癌5/5,低分化鼻咽癌4/4,何杰金氏病5/5,非何杰金氏病0/2,头颈其他肿瘤1/6,鼻咽慢性炎症0/5,正常鼻咽组织0/4;新生上皮细胞DNA抽提物;低分化鼻咽癌2/2,炎症0/1,正常人胚鼻咽上皮0/1;携带EBV基因组细胞系(Raji,B_(95-8)各1)2/2,致淋巴细胞转化之B_(95-8)病毒为10~(-4),PCR检测10~(-4)～10~(-6)均阳性,10~(-7)未检出。结果表明EBV与鼻咽癌与何杰金氏病有关,常规石蜡包埋切片仅8μm×0.1mm~2,贮存时间至三年仍可用于PCR检测EBV DNA,证实PCR是一种快速、灵敏和特异测捡EBV基因组的方法,可作为肿瘤和疚病病毒病因回顾性调直研究的有力手段。     

肾综合征出血热病毒基因检测及分型的研究

根据流行于我国的两型ＨＦＲＳＶ代表株汉滩型７６１１８株及汉城型Ｒ２２株Ｍ节段的核酸序列,设计两型共同引物,建立了逆转录－聚合酶链反应（ＲＴＰＣＲ）方法,检测３９株从不同地区、不同宿主分离的ＨＦＲＳＶ感染鼠脑及细胞培养物;同时还建立了捕捉ＥＬＩＳＡ法（ｃＥＬＩＳＡ）,检测了３９株中的３６株,每份样本设复孔,以Ｐ／Ｎ≥２．１０且Ｐ－Ｎ≥０．１０者判为阳性。ＲＴＰＣＲ及ｃＥＬＩＳＡ两法的检出率分别为９７．６％与８２．４％,二者符合率８４．６％。此外,对ＲＴＰＣＲ产物进行酶切分型,３８份扩增产物中的１５份可被ＡｌｕＩ切开。根据所获酶切图谱的差异,可分为汉滩型及汉城型两型,显示了酶切分型的潜在价值     

实验犬布氏杆菌的多重PCR检测与分型鉴定

目的建立实验犬及相关生物制品布氏杆菌的多重PCR检测与分型鉴定方法。方法选择布氏杆菌Omp2基因同源性较高的区域设计引物对布氏杆菌进行多重PCR扩增,扩增结果一致的样本进行酶切以区分不同型,同时进行序列测定,以确定该方法的准确性;然后验证该方法的特异性和敏感性。结果成功扩增得到目的条带,并通过酶切区分五种布氏杆菌;PCR产物与布氏杆菌DNA序列同源性达到99%,并验证了该方法的检测结果。实验结果证明该方法特异性较好,灵敏性为1.8×10^-7μg/mL。结论成功建立布氏杆菌多重PCR检测与分型鉴定方法,所建立的方法特异性好,灵敏度高。本研究对保证实验犬群的质量,保护饲养人员、实验人员的身体健康具有重要意义。     

Polymerase Chain Reaction for the Detection of Chlamydia psittaci in the Feces of Budgerigars

The polymerase chain reaction (PCR) targeting the ompA gene of Chlamydia psittaci was evaluated for its ability to detect chlamydiae in fecal specimens of budgerigars as compared with isolation procedures using cell culture and embryonated egg inoculations. Several procedures for PCR template DNA preparation were compared so as to determine their detection levels for chlamydiae propagated in cell culture in the presence of fecal materials. Tween-20 and proteinase K treatments followed by centrifugation of the template DNA were found to be an appropriate procedure for DNA preparation for primary PCR. Subsequent nested PCR was shown to detect 4.8 IFU/ml or 84 particles/ml of chlamydiae. Chlamydiae in 50 fecal specimens from apparently healthy budgerigars were examined by nested PCR and several other methods. Nested PCR detected chlamydiae at a higher rate (12/50, 24%) than the isolation procedure in embryonated eggs (6/50, 12%). Primary PCR combined with the isolation procedure in cell culture gave a detection rate (5/50, 10%) similar to that of isolation from embryonated eggs. Detection rates by primary PCR (1/50, 2%) and in cell culture (0%) were inferior to the other procedures.     

应用一步PCR法检测并鉴定马疱疹病毒1型和4型

聚合酶链反应（PCR）可作为一种快速检测并鉴别马疱疹病毒1型（EHV－1）和马疱疹病毒4型（EHV－4）的诊断方法。PCR引物是根据编码EHV－1和EHV－4的糖蛋白B（ gB)基因上的共有的核苷酸序列或型特有的核苷酸序列设计的。利用这两种病毒的型特异性混合引物,在一步PCR反应中检测并鉴别EHV－1和EHV－4,而同一疱疹病毒科的单纯疱疹病毒1型（HSV－1）、伪狂犬病毒（PRV）、马立克氏病病毒（MDV）均无特异性扩增。应用建立的PCR方法检测了普氏野马流产胎儿病科,实验结果表明,这种PCR方法是一种直接检测并鉴定EHV－1和EHV－4的快速、敏感的诊断方法,同时,它可在一步PCR反应中直接鉴别这两种病毒,可用于病料中EHV－1和EHV－4检测的初步筛选。