Specific Detection of Buckwheat Residues in Processed Foods by Polymerase Chain Reaction

A specific and qualitative detection method for buckwheat in foods using the polymerase chain reaction (PCR) was developed. Trace amounts of buckwheat in commercial food products were qualitatively detected by this method. It should be reliable for detecting buckwheat residues in processed foods and practical for monitoring the labeling system for allergenic food materials.     

Specific Discrimination of Chicken DNA from Other Poultry DNA in Processed Foods Using the Polymerase Chain Reaction

In the present study, specific discrimination of chicken DNA contamination in processed foods using the polymerase chain reaction was investigated. The primer pair was designed to amplify a 102-bp fragment of the chicken mitochondrial 16S ribosomal RNA gene. While the DNA from chicken meat was amplified, the DNA from other poultry meat, mammalian meat, fish, shellfish, and cereals was not amplified. The primer amplified DNA fragments derived from model processed and nonprocessed food samples containing 0.001, 0.01, 0.1, 1, 10, and 100% chicken.     

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。     

巢式甲基化特异性PCR检测肺癌病人WIF-1基因启动子区异常甲基化

为了检测肺癌患者血浆中WIF-I基因启动子区的甲基化状态,收集肺癌患者及健康对照者的血浆标本,采用巢式甲基化特异性PCR（nMSP）法检测WIF-I基因启动子区甲基化状态,并与普通甲基化特异性PCR（MSP）法进行了比较,结果在58例肺癌患者血浆样品中经nMSP法发现20例WIF-I基因启动子的过甲基化,用MSP法只检出10例,有吸烟史组WIF-1基因的甲基化率高于无吸烟史组（P〈0．05）．而20例正常对照血浆中都未检测到形胆J基因启动子的过甲基化;表明利用巢式MSP（nMSP）法检测外周血血浆中WIF-1基因启动子的甲基化,可为非损伤性筛选和早期诊断肺癌提供有价值的信息．     

Detection of Various Variant Verotoxin Genes in Escherichia coli by Polymerase Chain Reaction

We constructed common primers for the polymerase chain reaction to detect the genes for various Verotoxins reported, that is, VT1 (or SLT-I), VT2 (or SLT-II), VT2vha, VT2vhb, SLT-IIv (or VT2vp1, VTe) and SLT-IIva (or VT2vp2). A total of 80 Verocytotoxin-producing Escherichia coli strains isolated from humans, domestic animals and meats gave a positive result by PCR with the designed common primers. Digestion by restriction endonucleases BglII and EcoT14I of the amplicon of the VT2vp2 gene gave specific bands of the expected sizes, but not of the amplicons of other VT genes, suggesting a possible method for identification of the VT2vp2 gene. Application of the PCR with the designed primers in diagnostic and epidemiological studies on VTEC infection is also discussed.     

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%.     

巢式甲基化特异性PCR检测不同类型组织标本中WIF-1基因启动子异常甲基化

目的:采用一种高灵敏度的DNA甲基化分析方法,即巢式甲基化特异性PCR法(nested-MSP,nMSP),检测外科手术切除新鲜组织、石蜡包埋组织及纤维支气管镜活检组织中WIF-1基因启动子的异常甲基化状态。方法:将基因组DNA变性成为单链,用亚硫酸氢盐修饰单链DNA,所有未甲基化的胞嘧啶被转变为尿嘧啶,而甲基化的胞嘧啶则不变。设计针对甲基化和非甲基化等位基因的特异引物,进行巢式PCR扩增,最后经凝胶电泳检测目的片段。结果:在3种类型的原发性非小细胞肺癌标本中都检测出了WIF-1基因启动子的异常甲基化。结论:巢式甲基化特异性PCR是一种灵敏度高、特异性强的甲基化检测方法,可广泛应用于不同类型标本基因启动子甲基化的分析。     

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.     

Detection of Puccinia striiformis in Latently Infected Wheat Leaves by Nested Polymerase Chain Reaction

Stripe rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. The identification of the pathogen in infected plants based on morphological or physiological criteria before sporulation is labour-intensive and time-consuming. To accelerate and simplify the process of detection, a nested Polymerase Chain Reaction (PCR) assay was developed for specific and sensitive detection of Pst . Specific primers Psta-Psts were designed according to a genome-specific sequence of Pst . In nested PCR, with a 10-fold dilution series of template DNA, the detection limit was 2 pg DNA in the first PCR with the primers Psta-Psts. The second round PCR was then performed using amplified products from the first PCR as the template and Nesta-Nests as the primers. An amplification signal was detectable even when only 2 fg of P. striiformis f. sp. tritici DNA was used as the template in nested PCR. With nested PCR, the sensitivity of detection was enhanced 1000 fold. Using extracts from stripe rust-infected wheat leaves, the fungus could be determined in the leaves before symptom appearance. The assay provides a rapid and sensitive method for detection of P. striiformis f. sp. tritici in latently infected leaves of overwintering wheat plants.     

Detection of the protozoan Neospora caninum Using in situ Polymerase Chain Reaction

A new method to detect the protozoan Neospora caninum using indirect in situ polymerase chain reaction (PCR) is described. In situ PCR combines the advantages of the extraordinarily high sensitivity and specificity of PCR and the in situ representation of immunohistochemical methods. We describe an indirect in situ PCR, whereby the amplified products were detected using a primed in situ (PRINS) reaction with hapten-labeled nucleotides and visualized using fluorochrome-labeled antibodies. This technique was carried out in both infected cell cultures and formalin fixed, paraffin embedded tissues. Clear signals were obtained in the N. caninum positive samples using in situ PCR, whereas control slides with Toxoplasma gondii infected tissues always yielded negative results.     

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     

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     

荧光定量PCR技术在临床微生物检测中的应用

荧光定量PCR技术是近些年来发展起来的一种核酸检测技术,它以灵敏度高、特异性强、重复性好、快速准确定量等特点被广泛应用于各个领域。现主要介绍荧光定量PCR技术在病毒、细菌、真菌和寄生虫4个临床微生物检测方面的应用进展。     

用聚合酶链式反应（PCR）检测马铃薯纺锤块茎类病毒

用DNA合成仪合成两个马铃薯纺锤块茎类病毒(Potato spindle tuber viroid, PSTVd)特异性引物,从感病的马铃薯块茎组织的核酸抽提液中,用反转录酶合成PSTVd eDNA,然后用PCR法进行扩增,扩增产物用电泳检测,建立了用PCR法检测PSTVd的新方法。结果表明,该方法特异性强,灵敏度可达0.15pg,比现有其它检测方法高,而且样品用量少。     

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。     

聚合酶链式反应快速鉴定啤酒有害菌研究

建立了PCR快速鉴定啤酒有害菌的新方法。用基于抗酒花基因horA部分序列的特异引物对啤酒污染乳酸菌进行PCR检测,灵敏度可达到3个细胞(CFU),样品的预培养需12~16h。啤酒有害菌检测所需时间从传统的5d减少到24h。     

Specific Detection of Pacific Oyster (Crassostrea gigas) Larvae in Plankton Samples Using Nested Polymerase Chain Reaction

Management of sustainable Pacific oyster fisheries would be assisted by an early, rapid, and accurate means of detecting their planktonic larvae. Reported here is an approach, based on polymerase chain reaction (PCR), for the detection of Pacific oyster larvae in plankton samples. Species-specific primers were designed by comparing partial mitochondrial cytochrome oxidase subunit I (COI) sequences from Crassostrea gigas, with other members of the family Ostreidae including those of Crassostrea angulata. Assay specificity was empirically validated through screening DNA samples obtained from several species of oysters. The assay was specific as only C. gigas samples returned PCR-positive results. A nested PCR approach could consistently detect 5 or more D-hinge-stage larvae spiked into a background of about 146 mg of plankton. The assay does not require prior sorting of larvae. We conclude that the assay could be used to screen environmental and ballast water samples, although further specificity testing against local bivalve species is recommended in new locations.     

Specific Detection of Bjerkandera adusta by Polymerase Chain Reaction and Its Incidence in Fungus-Associated Chronic Cough

Chronic cough is a common symptom at outpatient care. An uncontrollable cough with difficulty in treatment is called chronic idiopathic cough. Recent reports have demonstrated that the presence of basidiomycetous fungi in sputum is an important clinical finding that assists in clarifying the cause of chronic cough in some cases. Research has suggested that Bjerkandera adusta is related to fungus-associated chronic cough (FACC). FACC is defined as a chronic cough associated with basidiomycetous fungi found in induced sputum and can be treated with antifungal medication. B. adusta is one of the basidiomycetous fungi that exist in cosmopolitan environments. The aim of this study was to develop a B. adusta detection method using polymerase chain reaction (PCR) with a specific primer set and to research the incidence of B. adusta in FACC. The new method successfully detected B. adusta from FACC patients. The incidence of B. adusta in FACC was 42.86 %. Antifungal drugs were effective in most cases. Significant differences in treatment duration between B. adusta patients and non-B. adusta patients were observed. It is therefore suggested that the presence of B. adusta may be one of the allergic intractable factors of chronic cough. This finding may provide identifiable differences in clinical manifestations between B. adusta and non-B. adusta in FACC and lead to possible differing remedies to treat the two forms. PCR can specifically detect B. adusta from patients suffering from chronic cough and provides a new diagnosis for FACC associated with B. adusta.