Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

新疆绵羊种布鲁氏菌HtrA基因的克隆与序列分析

根据已发表的牛流产型布鲁氏菌(B.abrotus)HtrA(High temperature requirment A)基因的核酸序列设计引物,从新疆绵羊种布鲁氏菌(B.ovis)基因组中扩增到了大约1600bp的片段。将该片段纯化后克隆到PBS-T载体上,对所得到的重组质粒进行PCR鉴定、酶切分析后,对克隆的片段进行测序表明,新疆绵羊种布鲁氏菌HtrA基因与发表的牛流产型布鲁氏菌、马耳他布鲁氏菌(B.melitensis)、猪种布鲁氏菌(B.suis)的HtrA基因核苷酸序列的同源性分别为99.68%、99.81%、99.55%,推导的氨基酸序列也存在很高的同源性。     

布鲁氏菌2308ery基因启动子缺失株的构建及鉴定

目的:构建布鲁氏菌2308株ery基因启动子缺失株。方法:用PCR方法从亲本株2308上扩增ery基因启动子侧翼序列,将该片段与pMD19-T连接,亚克隆为自杀载体pGEM-7zf-Δery-sacB。将自杀载体电转化布鲁氏菌感受态细胞中经同源重组后,分别用100 mg/L氨苄和7%蔗糖筛选。对获得的基因缺失株进行RT-PCR鉴定和遗传稳定性检测。结果:成功获得ery基因启动子缺失株,2308Δery基因启动子缺失株未扩增出eryA基因。并且该缺失株在10代以内未发生回复突变。结论:成功构建2308Δery基因启动子缺失株,为研究布鲁氏菌的毒力基因及其流产机制奠定基础。     

Comparison of three different PCR methods for detection of Brucella spp in human blood samples

In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i). a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii). a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii). a gene encoding an outer membrane protein (omp-2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.     

The use of an arbitrarily primed PCR product for the specific detection of Brucella

A 1.3 kb Brucella-specific DNA fragment produced through the use of arbitrarily primed polymerase chain reaction (AP-PCR) was tested for its specificity by DNA–DNA hybridization to Brucella and non-Brucella bacteria. The digoxigenin (DIG)-labelled 1.3 kb DNA fragment hybridized with Brucella abortus and Brucella melitensis but did not hybridize with other non-Brucella bacteria tested. The sensitivity of the reaction was determined; as little as 150 fg DNA or 30 Brucella cells could be detected. The specificity and sensitivity of the 1.3 kb DNA fragment combined with the simplicity and speed of the technique suggest the potential of this fragment as a DNA probe for the quick and reliable detection of Brucella organisms.     

应用xMAP液态芯片多重快速检测四种病原微生物的研究

目的：建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法：根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果：xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论：xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。     

应用xMAP液念芯片多重快速检测四种病原微生物的研究

目的:建立一种多重、快速、特异性好、灵敏度高的病原微生物检测方法。方法:根据GenBank数据库中的小肠结肠炎耶尔森氏菌、单核细胞增生性李斯特菌、产气荚膜梭菌、鼠疫耶尔森氏菌基因序列,分别针对ail、hly、cpe、3a基因设计4对引物和4条探针。通过重叠PCR扩增各目的基因并构建重组质粒,以该重组质粒DNA为模板,通过多重PCR同时扩增上述4个基因,建立xMAP液态芯片检测技术,在此基础上对标准菌株基因组DNA进行检测并验证该方法的特异性和敏感性。结果:xMAP液态芯片对质粒DNA和标准菌株基因组DNA的检测结果与多重PCR结果一致。该方法能在3.5 h内同时完成对4种病原菌的检测,特异性好,且敏感性要高于PCR方法,灵敏度最高可达200CFU/ml。结论:xMAP液态芯片技术是病原微生物的多重快速检测的新方法,具有很好的应用价值和前景。     

A highly sensitive non-isotopic system of DNA hybridization using amplification (PCR) for identifying and indicating presence of Brucella]

A non-isotopic amplification system was used to identify and indicate Brucella. The terminal sequences of a protein gene fragment in Brucella outer membrane were identified and direct and reverse primers were chosen for a polymerase chain reaction. (PCR). PCR amplifies a specific DNA fragment, 700 kb in size, only in representatives of the Brucella genus. A probe was design, which is the central part of the amplified DNA fragment, 550 kb in size. Single Brucella cells were detectable with an unlabelled probe in the analyzed samples during hybridization reactions. The system can be recommended for a rapid and reliable analysis in medical and veterinary practice.     

Detection of a specifically amplified DNAfragment in Brucella abortus by arbitrarilyprimed polymerase chain reaction

Randomly amplified polymorphic DNA (RAPD) profiles of Brucella and non-Brucella DNA were established after polymerase chain reaction (PCR) amplification. Five arbitrary oligonucleotide primers were screened to generate Brucella-specific DNA fingerprints. The arbitrary primer OPB-01 (5-GTTTCGCTCC-3) produced DNA bands specific to Brucella. Amplification conditions must be optimized for reproductibility. Accordingly, we optimized and established the conditions, which included Mg²⁺, enzyme (DNA polymerase), primer, template and deoxyribonucleoside triphosphate (dNTP) concentrations as well as the optimum number of thermal cycles to produce OPB-01 directed Brucella DNA fingerprints.The optimized RAPD method can produce a 1.3 kb DNA fragment specific to Brucella. This DNA fragment was common to eight biovars of B. abortus and one biovar of B. melitensis. The fragment was not detected in genetically related species such as Ochrobactrum anthropi and other non-Brucella organisms associated with farm animals. We anticipate the use of this fragment as a possible probe for the detection of Brucella organisms.     

盘羊肺炎支原体感染PCR 检测方法的建立与初步应用

羊支原体性肺炎,是由支原体引起的一类高度接触性传染病,又称羊传染性胸膜肺炎(Infections pleuropneumonia of sheep and goats),临床症状主要表现为高热、咳嗽、消瘦、     

Rapid,sensitive detection of Brucella abortus by polymerase chain reaction without extraction of DNA

Summary A freeze-thaw method was used to release DNA from acetone-killed, dried Brucella abortus S19 cells for polymerase chain reaction. A 635 bp fragment of a 43 kd membrane protein gene was amplified and detected by agarose gel electrophoresis. The sensitivity of the detection was as little as 100 fg of cells/sample.     

Multiple displacement amplification in combination with high-fidelity PCR improves detection of bacteria from single females or eggs of Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae)

Amplifying microbial DNA by the polymerase chain reaction (PCR) from single phytoseiid mites has been difficult, perhaps due to the low titer of bacteria and to interference by the relatively larger amounts of mite genomic DNA. In this paper we evaluate the efficiency of standard and high-fidelity PCR protocols subsequent to amplification of the whole genome by a multiple displacement amplification (MDA) procedure developed by Dean et al. DNA from the phytoseiid Phytoseiulus persimilis (Athias-Henriot) was tested because it lacks a Cytophaga-like organism (CLO) and we could add known amounts of a plasmid containing a cloned 16S rRNA gene fragment from a CLO from Metaseiulus occidentalis (Nesbitt). P. persimilis genomic DNA was mixed with the serially diluted plasmid and amplified using MDA followed by either standard or high-fidelity PCR. MDA followed by high-fidelity PCR was most efficient and successfully amplified an expected 1.5-kb band from as little as 0.01fg of the plasmid, which is equivalent to about 1 copy. MDA followed by high-fidelity PCR also consistently amplified Wolbachia- or CLO-specific products from naturally infected single females or eggs of M. occidentalis, which will allow detailed studies of infection frequency and transmission of several microorganisms associated with this predatory mite.     

Loop-mediated isothermal amplification (LAMP) for rapid detection of Renibacterium salmoninarum, the causative agent of bacterial kidney disease

A loop-mediated isothermal amplification (LAMP) assay was developed for rapid, specific and sensitive detection of Renibacterium salmoninarum in 1 h without thermal cycling. A fragment of R. salmoninarum p57 gene was amplified at 63 degrees C in the presence of Bst polymerase and a specially designed primer mixture. The specificity of the BKD-LAMP assay was demonstrated by the absence of any cross reaction with other bacterial strains, followed by restriction digestion of the amplified products. Detections of BKD-LAMP amplicons by visual inspection, agrose gel electrophoresis, and real-time monitoring using a turbidimeter were equivalently sensitive. The BKD-LAMP assay has the sensitivity of the nested PCR method, and 10 times the sensitivity of one-round PCR assay. The lower detection limit of BKD-LAMP and nested PCR is 1 pg genomic R. salmoninarum DNA, compared to 10 pg genomic R. salmoninarum DNA for one-round PCR assay. In comparison to other available diagnostic methods, the BKD-LAMP assay is rapid, simple, sensitive, specific, and cost effective with a high potential for field application.     

Rapid identification of Lactobacillus brevis using the polymerase chain reaction

AIMS: Species-specific PCR was applied to identify Lactobacillus brevis and the sensitivity and the specificity of the protocol were determined. METHODS AND RESULTS: Strains of Lact. brevis obtained from foods, particularly dairy products, and various strain collections, were identified by PCR using primers which amplified a 1340 bp fragment within the 16S rRNA gene. The PCR product was obtained after amplification of all the Lact. brevis strains tested; the size of the amplicon was as expected. No PCR products were observed after amplification from DNA of several lactic acid bacteria (LAB) species. CONCLUSIONS: A PCR method was optimized to identify Lact. brevis. The protocol was highly efficient and sensitive. SIGNIFICANCE AND IMPACT OF THE STUDY: Conventional phenotypic methods often lead to ambiguous identification of LAB species belonging to Lact. brevis. The proposed protocol is sensitive, specific, and can be applied to total DNA extracted by use of chelating matrix with loss of neither sensitivity nor specificity.     

Rapid detection of Brucella spp. by the loop-mediated isothermal amplification method

Aims:  To develop a rapid and sensitive method for detecting Brucella spp.
Methods and Results:  Two sets of six Brucella -specific primers for loop-mediated isothermal amplification (LAMP) were designed from the sequence of the Brucella abortus BCSP31 gene. The specificity and sensitivity were examined for six Brucella species (22 strains) and 18 non- Brucella species (28 strains). The LAMP assay was specific to Brucella spp. in 35 min at 63°C and sensitive (detected 10 fg of genomic DNA). The assay was also applied for the detection of Brucella DNA in contaminated milk and infected mouse organs.
Conclusions:  We developed a sensitive and specific LAMP assay for Brucella spp., with the test appearing to be useful for the detection of the pathogen from clinical and food samples.
Significance and Impact of the Study:  This is the first report of the development of LAMP for the detection of Brucella spp. As the LAMP assay can be performed at a constant temperature and its reactivity is directly observed with the naked eye without electrophoresis, our assay should be useful for the diagnosis of brucellosis as well as the detection of the bacteria in environmental or food samples.     

肺炎支原体P1蛋白羧基端基因片段在大肠杆菌中克隆的研究

在大肠杆菌中克隆肺炎支原体P1蛋白羧基端基因片段,为P1蛋白基因片段的扩增、表达及探讨羧基端基因片段功能打基础.采用PCR扩增方法获取P1结构基因.扩增产物用SalI和EcoRI酶切消化,回收1kb大小的DNA片段并与pUC19DNA连接,转入大肠杆菌JM109菌株.用X-gal平板及质粒图谱分析方法筛选重组克隆株,再用限制性核酸内切酶酶切图谱分析鉴定.经PCR扩增MPDNA获得1条5.0kbDNA片段.重组质粒限制性内切酶指纹图谱显示出2条带,1条为pUC19载体DNA带,另1条是1kb的插入片段.实验获得肺炎支原体P1蛋白结构基因及含P1蛋白羧基端DNA片段的重组克隆株.     

Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species

AIMS: Nine sets of PCR primers targeting Salmonella were evaluated for their specificity with pure cultures of intestinal-associated bacteria prior to their application to Salmonella detection in faecal samples. METHODS AND RESULTS: Gene targets of PCR primers included: 16S rDNA, a Salmonella pathogenicity island I virulence gene, Salmonella enterotoxin gene (stn), invA gene, Fur-regulated gene, histidine transport operon, junction between SipB and SipC virulence genes, Salmonella-specific repetitive DNA fragment, and multiplex targeting invA gene and spvC gene of the virulence plasmid. Fifty-two Salmonella strains were used to determine sensitivity; five strains from related genera and 45 intestinal bacteria were used to evaluate specificity. All primers amplified DNA from Salmonella strains, although two primer sets failed to amplify Salmonella DNA from either Salmonella bongori (hilA) or subgroups VI or VII (16S rDNA). There was no detected amplification of DNA from related bacterial genera with any of nine PCR assays. Six of the PCR assays amplified DNA for some intestinal bacteria. CONCLUSIONS: Only three primer pairs were determined to be suitable for application of PCR amplification of Salmonella in faecal samples - 16S rDNA, stn and histidine transport operon. We are currently evaluating their sensitivity of detection of Salmonella in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the importance of internal lab validation of PCR primers prior to application to the type of samples of interest. Information from this evaluation can be applied in other labs to facilitate choosing Salmonella PCR primers.     

A polymerase chain reaction-based method to detect cisplatin adducts in specific genes.

Every bulky lesion in DNA can potentially inhibit the Taq DNA polymerase and thereby decrease the amplification produced in the polymerase chain reaction. We investigated the feasibility of using this inhibition to quantify DNA lesions produced by the anticancer drug cisplatin. Products were detected by electrophoresis followed by ethidium bromide staining. Quantitation was obtained by including [32P]dCTP in the amplification reaction and subsequently assessing the incorporated radioactivity. Hamster genomic DNA was platinated in vitro to defined levels and amplified with primers that produce either a 150, 750 or 2,000 base pair fragment. The degree of inhibition of PCR agreed with the predicted level of DNA platination in each size of fragment, suggesting that the polymerase was inhibited by every cisplatin-induced lesion. This method was used to detect cisplatin-induced lesions in the adenine phosphoribosyltransferase gene of CHO cells. Cells were incubated with 0-125 microM cisplatin for 2 h, the DNA was purified and subjected to PCR. A significant decrease in amplification of the 2 kbp fragment was observed in DNA from cells incubated with cisplatin at 75 microM. The degree of inhibition agreed closely with the amount of DNA damage in the overall genome as measured by atomic absorption. No change was detected in amplification of the 150 base fragment which can therefore be used to normalize data for any variations between DNA samples. This assay has the same sensitivity as other methods currently used for the analysis of gene-specific damage. The advantage of this assay is that it obviates the need for specific endonuclease complexes to recognize and cleave DNA adducts as previously required when analyzing damage in specific genomic sequences.     

牛分枝杆菌mpb64基因的克隆、鉴定及其表达

以牛型分枝杆菌基因组DNA为模板,PCR方法扩增mpb64基因,纯化PCR产物并与pDM18-T载体连接、转化,经酶切及核苷酸序列鉴定为正确后,酶切产物亚克隆到原核表达载体pET30a(+)的KpnI／EcoRI位点,构建重组表达质粒pET30a+-mpb64,转化到大肠杆菌DE3内,以IPTG进行诱导,终浓度为1mmol／L,诱导产物进行SDS-PAGE电泳。结果表明,PCR方法成功扩增出mpb64基因,核苷酸序列测定验证了其正确性,重组表达质粒表达的pET30a+-mpb64融合蛋白相对分子量为30.4kDa,与实测相符。牛分枝杆菌pET30a+-mpb64的成功表达为牛结核病的诊断及新型疫苗的研究奠定了基础。     

Re-usable DNA template for the polymerase chain reaction.

DNA covalently bound to an uncharged nylon membrane was used for consecutive amplifications of several different genes by PCR. Successful PCR amplifications were obtained for membrane-bound genomic and plasmid DNA. Membrane-bound genomic DNA templates were re-used at least 15 times for PCR with specific amplification of the desired gene each time. PCR amplifications of specific sequences of p53, p16, CYP1A1, CYP2D6, GSTM1 and GSTM3 were performed independently on the same strips of uncharged nylon membrane containing genomic DNA. PCR products were subjected to restriction fragment length polymorphism analysis, single-strand conformational polymorphism analysis and/or dideoxy sequencing to confirm PCR-amplified gene sequences. We found that PCR fragments obtained by amplification from bound genomic DNA as template were identical in sequence to those of PCR products obtained from free genomic DNA in solution. PCR was performed using as little as 5 ng genomic or 4 fg plasmid DNA bound to membrane. These results suggest that DNA covalently bound to membrane can be re-used for sample-specific PCR amplifications, providing a potentially unlimited source of DNA for PCR.