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.     

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.     

Polymerase Chain Reaction Test for Differentiation of Five Toxin Types of Clostridium perfringens

In order to avoid the use of experimental animals, the polymerase chain reaction (PCR) method was applied to differentiate Clostridium perfringens into five toxin types. Twenty-two out of 23 strains tested produced the toxin(s) corresponding to the toxin gene(s) identified by PCR, and vice versa. Consequently, the gene typing was consistent with conventional typing by animal tests. Twenty-five strains were identified as types different from original ones by the PCR method as well as a toxin neutralization test. These findings suggest that the PCR method, which is easy and timesaving, is applicable to identify the toxin types of C. perfringens as an alternative to animal tests, and that beta-, epsilon- and iota-toxin genes might be lost by long-term preservation. The reasons why the strains lost the genes are discussed.     

Detection and Identification of Bursaphelenchus Species with DNA Fingerprinting and Polymerase Chain Reaction

We have evaluated the potential of DNA-based methods to identify and differentiate Bursaphelenchus spp. and isolates. The isolation of a DNA probe, designated X14, and development of a DNA fingerprinting method for the identification and differentiation of Bursaphelenchus species and strains is described. Polymerase chain reaction (PCR) amplification of DNA isolated from Bursaphelenchus species using two primers derived from the sequence of the cloned repetitive DNA fragment X14 resulted in multiple band profiles. A 4-kb fragment thus amplified from B. xylophilus DNA was not amplified from B. mucronatus or B. fraudulentus DNA. In addition to this fragment, several other fragments are amplified from the three species. The banding patterns obtained allowed species identification and may have value in determining taxonomic affinities.     

Molecular Characterization of Human Rotavirus VP4 Genes by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Assay

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic variability and similarity of the VP4 genes of human rotaviruses. The VP4 genes of 14 human rotavirus strains, including VP4 serotype P1A strains (Wa, P, VA70), serotype P1B strain (DS-1), serotype P2 strains (M37, 1076, McN, ST3) and serotype P3 strains (AU-1, AU228, K8, PA151, PCP5, MZ58), and those of 2 feline strains (FRV-1 and Cat2) were reverse-transcribed and amplified by the polymerase chain reaction (PCR). The amplified VP4 cDNAs were then digested with a panel of restriction endonucleases (HindIII, NruI, HaeIII, and EcoRI), resulting in the identification of at least one enzyme with which digestion produced an RFLP profile specific for a particular P serotype. Of interest was the presence of two distinct RFLP patterns within the serotype P3 VP4 genes: one corresponding to the VP4 gene carried by the members of the AU-1 genogroup and the other corresponding to the VP4 genes carried by naturally-occurring reassortants between members of the AU-1 and other genogroups.     

Use of Polymerase Chain Reaction and Restriction Enzyme Analysis to directly detect and identify Salmonella typhimurium in food

A primer set of oligonucleotides (Salm 3 and Salm 4) from the inv A gene of Salmonellae has been evaluated for the specific detection of Salmonella spp. by the polymerase chain reaction (PCR). This primer set amplified 33 Salmonella serovars but did not amplify 16 non- Salmonella bacteria. Moreover, after PCR amplification, it was possible to identify Salm. typhimurium by restriction enzyme analysis. The PCR-RE method developed could represent a helpful tool for detecting Salmonella spp., and for directly and rapidly identifying Salm. typhimurium in food.     

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。     

聚合酶链反应筛选阳性重组体

聚合酶链反应（ＰＣＲ）技术已广泛用于ＤＮＡ基础研究和临床诊断。本文介绍用它来鉴定靶序列,从而进一步筛选阳性重组体。由于引物Ｔｍ较高,采用二温段扩增法,确实扩增出１０ｂｐ靶序列。文章还讨论了扩增较小片段要注意的条件。     

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。     

Differentiation of Xanthomonas campestris pvs. pelargonii and hederae by Polymerase Chain Reaction

Geranium isolates of Xanthomonas campestris pv. pelargonii ( Xcp ) and English ivy isolates of X. campestris pv. hederae ( Xch ) were tested by polymerase chain reaction (PCR) to determine whether the two pathovars could be discriminated using amplification conditions developed to identify and detect Xcp in infected geraniums. Using PCR, other workers reported that the genomes of Xcp and Xch were indistinguishable. The objective of this study was to determine whether the two pathovars have sufficient sequence diversity to allow them to be distinguished by molecular means. Three primer pairs were used for PCR amplification. Two of the primer pairs (REP and XcpM1/XcpM2) were able to distinguish between Xch and Xcp , whereas amplification with the third primer pair (ERIC) did not allow discrimination between the two pathovars. Based on PCR amplification, Xcp and Xch are distinctly different pathovars. Additionally, all three primer pairs showed discrimination between Xcp and Acidovorax , a bacterial pathogen that induces leaf spot on geranium.     

用PCR法直接快速筛查重组阳性克隆

本研究目的是应用PCR法快速筛查插入有苯丙氨酸脱氨酶（PAL）cDNA重组阳性克隆,用于PCR扩增的引物是位于载体pET23b启动子处的T7启动子引物和位于目的基因PALc DNA3’端终止密码TAA处的引物,以灭菌吸头挑一单菌落加PCR体系扩增。结果：在筛查的3个克隆中,有2个阳性克隆,并且插入方向正确,经DNA序列测定得到进一步证实,结果表明,以PCR方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方向,不需提取质粒。     

乙型肝炎病毒和丙型肝炎病毒的同步PCR扩增

介绍了一种从人血清中同步扩增和检测ＨＢＶ－ＤＮＡ和ＨＣＶ－ＲＮＡ的方法。ＨＣＶ－ＲＮＡ反转录成ｃＤＮＡ,这种ｃＤＮＡ和从ＨＢＶ中抽提出的ＤＮＡ一起,用根据ＨＢＶ、ＨＣＶ保守区序列设计的特异引物进行同步ＰＣＲ扩增,这种方法对于检测ＨＢＶ和ＨＣＶ重复感染很有用处     

Sperm Mediated Transfer of Genes into Goldfish and Detection by Polymerase Chain Reaction

Goldfish sperms were mixed with eggs for fertilization after incubation with antifreeze protein gene(AFP)from ocean pout for 30 min.A number of embryos and 145 adult goldfish were obtained.DNA from adult goldfish and embryos was extracted separately.Results of the amplification by PCRand Southern blot molecular hybridization indicate the integration of exogenous antifreeze gene intothe genome of a part of the recipient goldfish.Of the 45 samples detected by PCR,twelve showedpositive reaction with distinct hybridization band.The positive rate was 26%.     

Equine Herpesviruses 1 and 4: Amplification and Differentiation by Polymerase Chain Reaction

Equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are important pathogens responsible for considerable economic losses in the horse industry. Differentiation between these 2 viruses using conventional serological methods with polyclonal antisera has been difficult. Biological differences have, however, been recognised for a long time. Both EHV-1 and EHV-4 are associated with upper respiratory disease, but disseminated infection with EHV-1 can result in neurological disorders or abortion in susceptible mares.     

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。     

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。     

Variation Within and Between Phytophthora Species from Rubber and Citrus Trees in China, Determined by Polymerase Chain Reaction Using RAPDs

Variation among 39 isolates of Phytophthora of six morphological species (P. citrophthora. P. parasitka, P. capsici, P. palmivora and P. meadii. from rubber and citrus trees, and P. colocasiae from taro) was studied using random amplified polymorphic DNA (RAPD) analysis. Ten randomly-chosen 10-mer primers were used. Generally, the banding patterns were similar within species and different between species, but no one primer was able to distinguish all six species from one another. Cluster analysis on pooled data from all the primers gave six groups of isolates corresponding to the six morphological species. The group corresponding to P. citrophthora was divided further into subgroups that were related to host species and geographical location. This work confirmed the existing morphological classification of Phytophthora isolates from rubber and citrus trees in tropical China and showed the validity of using RAPDs to study the taxonomy of Phytophthora.     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒(CAV)环形基因组DNA(全长2.3kb)的EcoRI位点和BamHI位点的两侧选择适当序列合成两对引物,用PCR技术,从斑点杂交检测到病毒核酸的CAV感染的MDCC-RP1细胞基因组DNA中,分别扩增出包含EcoRI和BamHI分割开的病毒基因组两部分(1.5kb和0.8kb)约1.5kb和约1.25kb的两个片段.再将其中相应序列拼接克隆进pUC18载体,获得包含CAV全基因组序列DNA片段的克隆质粒pCAV2.4.酶切分析表明,该质粒具有预期的BamHI位点、PstI位点、HindⅢ位点,而预期的EcoRI位点消失.重组质粒插入DNA片段的两端序列分析表明,质粒pCAV2.4是包含CAV全基因组序列的重组质粒,插入DNA片段序列中的EcoRI位点序列发生了一个碱基突变.     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒（ＣＡＶ）环形基因组ＤＮＡ（全长２．３ｋｂ）的ＥｃｏＲＩ位点和ＢａｍＨＩ位点的两侧选择适当序列合成两对引物,用ＰＣＲ技术,从斑点杂交检测到病毒核酸的ＣＡＶ感染的ＭＤＣＣＲＰ１细胞基因组ＤＮＡ中,分别扩增出包含ＥｃｏＲＩ和ＢａｍＨＩ分割开的病毒基因组两部分（１．５ｋｂ和０．８ｋｂ）约１．５ｋｂ和约１．２５ｋｂ的两个片段。再将其中相应序列拼接克隆进ｐＵＣ１８载体,获得包含ＣＡＶ全基因组序列ＤＮＡ片段的克隆质粒ｐＣＡＶ２．４。酶切分析表明,该质粒具有预期的ＢａｍＨＩ位点、ＰｓｔＩ位点、ＨｉｎｄⅢ位点,而预期的ＥｃｏＲＩ位点消失。重组质粒插入ＤＮＡ片段的两端序列分析表明,质粒ｐＣＡＶ２．４是包含ＣＡＶ全基因组序列的重组质粒,插入ＤＮＡ片段序列中的ＥｃｏＲＩ位点序列发生了一个碱基突变。     

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