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.     

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.     

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 60 degrees C. 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.     

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.     

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。     

Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes.

The 16S-23S rRNA spacer regions of Brucella abortus, B. melitensis, and B. suis were cloned and subcloned after PCR amplification. Sequence analysis of the inserts revealed a spacer of about 800 bp with very high ( > 99%) homology among the three species examined. Two genus-specific primer pairs, BRU-P5-BRU-P8 and BRU-P6-BRU-P7, that could be used in a nested PCR format and three genus-specific DNA probes, BRU-ICG2, BRU-ICG3, and BRU-ICG4, were deduced from this spacer. The specificity and sensitivity of both primer sets and probes were examined by testing them against a collection of 18 Brucella strains and 56 strains from other relevant taxa by using PCR and the Line Probe Assay (LiPA), respectively. A method for direct detection of Brucella spp. in 1 ml of raw milk was developed on the basis of enzymatic treatment of the milk components and subsequent PCR and LiPA hybridization. After a single PCR, sensitivities of 2.8 x 10(5) and 2.8 x 10(4) CFU/ml were obtained for detection by agarose gel electrophoresis and LiPA, respectively. Nested PCR yielded a sensitivity of 2.8 x 10(2) CFU/ml for both methods.     

Highly sensitive enzyme-linked assay based on monoclonal antibodies for detection of Brucella antigens

Mice monoclonal antibodies against lypopolysaccharides (LPS) of Brucella abortus has been obtained and characterized. The antibodies detected LPS of B. abortus, B. melitensis and B. suis with high sensivity and specificity and did not react with LPS of Yersinia enterocolitica O:3, Y. enterocolitica O:9, Salmonella typhimurium, and Francisella tularensis. It has been shown that interaction of monoclonal antibodies and LPS of Brucella species can be critically dependent from buffer system. Obtained monoclonal antibodies allowed to develop highly sensitive assay which was able to detect antigens of Brucella species in concentrations 0.05 - 0.1 ng/ml. The assay can be used for detection and identification of Brucella species.     

布氏杆菌病疫苗的应用和研究现状

布氏杆菌病是由布氏杆菌引起的一种重要的人兽共患病。布氏杆菌具有宿主广泛、传染性强以及感染后根治困难等特点,对畜牧业和人类健康均构成严重威胁,疫苗免疫是预防和控制布氏杆菌病的主要措施。迄今国内外已有多个弱毒活疫苗在使用,但均存在一定的缺陷,因此研究更理想的疫苗一直是控制布氏杆菌病的重点。目前除了常规诱变筛选新的弱毒株外,人们正通过基因工程技术构建重组弱毒疫苗、DNA疫苗以及亚单位疫苗。本文简述了布氏杆菌病疫苗的应用及新型疫苗的研究现状。     

Detection and differentiation of the six Brucella species by polymerase chain reaction.

BACKGROUND: Brucelosis is a severe acute febrile disease caused by bacteria of the genus Brucella. Its current diagnosis is based on clinical observations that may be complemented by serology and microbiological culture tests; however, the former is limited in sensitivity and specificity, the latter is time consuming. To improve brucelosis diagnosis we developed a test which is specific and sensitive and is capable of differentiating the six species of Brucella. MATERIALS AND METHODS: Four primers were designed from B. abortus sequences at the well-conserved Omp2 locus that are able to amplify the DNAs of all six species of Brucella. RESULTS: Our test detected all six species of Brucella. Their differentiation resulted directly from differences in the amplification patterns or was achieved indirectly using a RFLP present in one of the PCR products. The sensitivity and specificity of the new test were then determined; it was applied successfully in confirming the diagnosis of a patient whose clinical history and serology indicated infection with Brucella. CONCLUSIONS: The results make possible the use of a PCR test for Brucella detection and differentiation without relying on the measurement of the antibodies or microorganism culture. Our first results showed that the PCR test can confirm the presence of Brucella in blood samples of infected patients.     

Real-time PCR detection of Brucella abortus: a comparative study of SYBR green I, 5'-exonuclease,and hybridization probe assays

Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches-SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)-were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and the IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.     

Rapid combined assay for Salmonella detection in food samples

A rapid method was developed to detect salmonellae in food samples. The method gave a possibility to obtain results after 28 h 30 min. The preenrichment in buffered peptone water lasted for 6 h, the enrichment in Rappaport-Vassiliadis medium was applied for 18 h followed by PCR with INVA1-INVA2 primer pair, adapting Chiu and Ou's method. This procedure was suitable to demonstrate salmonella contamination at min. 10 cfu/25 g sample. Out of 18 samples there was a good agreement between the results of the conventional and rapid methods in case of 17 samples. PCR with SPVC1-SPVC2 primer pair informing about the presence of virulence plasmid was performed in separate tubes, because decreased sensitivity was observed in case of multiplex PCR.     

A polymerase chain reaction for detection of Brucella canis in vaginal swabs of naturally infected bitches

A PCR assay for the detection of Brucella canis in canine vaginal swab samples was evaluated, comparing its performance with that of bacterial isolation, serological tests, and a blood PCR assay. One hundred and forty-four female dogs were clinically examined to detect reproductive problems and they were tested by the rapid slide agglutination test, with and without 2-mercaptoethanol (2ME-RSAT and RSAT, respectively). In addition, microbiological culture and PCR were performed on blood and vaginal swab samples. The results of the vaginal swab PCR were compared to those of the other tests using the Kappa coefficient and McNemar test. Of the 144 females that were examined, 66 (45.8%) were RSAT positive, 23 (15.9%) were 2ME-RSAT positive, 49 (34.02%) were blood culture positive, 6 (4.1%) were vaginal swab culture positive, 54 (37.5%) were blood PCR positive, 52 (36.2%) were vaginal swab PCR positive, and 50.69% (73/144) were positive by the combined PCR. The PCR was able to detect as few as 3.8 fg of B. canis DNA experimentally diluted in 54 ng of canine DNA, extracted from vaginal swab samples of non-infected bitches. In addition, the PCR assay amplified B. canis genetic sequences from vaginal swab samples containing 1.0 x 10(0) cfu/mL. In conclusion, vaginal swab PCR was a good candidate as a confirmatory test for brucellosis diagnosis in bitches suspected to be infected, especially those negative on blood culture or blood PCR; these animals may be important reservoirs of infection and could complicate attempts to eradicate the disease in confined populations.     

TaqMan探针荧光PCR检测肠出血性大肠埃希菌O157:H7方法的建立

肠出血性大肠埃希菌(Enterohemorrhagie Escherichia coli,EHEC)O157:H7是一种重要的传染病病原菌,以EHEC O157:H7标准菌株rfbE保守区设计一对特异引物和一条探针,建立了检测EHEC O157:H7核酸的荧光定量PCR检测方法。实验结果表明荧光定量PCR检测方法特异性好,最低检测限为20 cfu/mL,线性范围是102～108cfu/mL。稳定性试验表明批内变异系数和批间变异系数分别为2.06%和2.45%。     

添加有扩增内标的副溶血弧菌PCR检测方法的建立

摘要：【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌（Vibrio parahaemolyticus）基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物（vp1332L/vp1332R）,同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343 bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499 bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102 cfu/mL。人工污染实验表明,起始染菌量为1.24 cfu/25 g样品时经8 h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。     

添加有扩增内标的副溶血弧菌PCR检测方法

【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌(Vibrio parahaemolyticus)基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物(vp1332L/vp1332R),同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102cfu/mL。人工污染实验表明,起始染菌量为1.24cfu/25g样品时经8h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。     

Genetic diversity of Desulfovibrio spp. in environmental samples analyzed by denaturing gradient gel electrophoresis of [NiFe] hydrogenase gene fragments.

The genetic diversity of Desulfovibrio species in environmental samples was determined by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified [NiFe] hydrogenase gene fragments. Five different PCR primers were designed after comparative analysis of [NiFe] hydrogenase gene sequences from three Desulfovibrio species. These primers were tested in different combinations on the genomic DNAs of a variety of hydrogenase-containing and hydrogenase-lacking bacteria. One primer pair was found to be specific for Desulfovibrio species only, while the others gave positive results with other bacteria also. By using this specific primer pair, we were able to amplify the [NiFe] hydrogenase genes of DNAs isolated from environmental samples and to detect the presence of Desulfovibrio species in these samples. However, only after DGGE analysis of these PCR products could the number of different Desulfovibrio species within the samples be determined. DGGE analysis of PCR products from different bioreactors demonstrated up to two bands, while at least five distinguishable bands were detected in a microbial mat sample. Because these bands most likely represent as many Desulfovibrio species present in these samples, we conclude that the genetic diversity of Desulfovibrio species in the natural microbial mat is far greater than that in the experimental bioreactors.     

全细胞多重PCR检测蓝藻、微囊藻及产毒微囊藻方法初探

选取三对分别针对微囊藻、蓝藻16S rDNA及微囊藻毒素合成酶基因mcyB的保守序列的特异性引物209F/409R、27F1/409R、MTR/MTF,其中409R为一条共用引物。设计并优化了一种可以同时检测蓝藻和微囊藻的两重全细胞PCR方法和一种可以同时检测蓝藻、微囊藻和可产毒微囊藻的三重全细胞PCR方法,并且测试了这两种PCR反应的灵敏度区间,分别为10⁵～10³cell·mL^-1、10⁵～10²cell·mL^-1。对采集水库水样检测结果表明双重全细胞PCR方法可以直接应用于对天然水样的检测,三重全细胞PCR方法可用于实验室培养藻细胞的筛查。全细胞多重PCR方法具有快速、简便、准确等特点,在水体微囊藻毒素检测预警方面具有应用价值。     

食品中沙门氏菌分子检测靶点的筛选与评价

[目的]发掘新的沙门氏菌分子检测靶点,筛选检测性能优秀的引物.[方法]利用BLAST程序比较沙门氏菌属内基因组DNA序列的同源性以及沙门氏菌与非沙门氏菌基因组DNA序列之间的特异性,发掘出100多个检测沙门氏菌属的特异性片段,并从中随机挑选出15个片段作为候选靶点,一共设计了27对引物(FS1～FS27),对它们的特异性、灵敏度加以评价,从中筛选检测性能最好的引物.[结果]在27对引物中,检测性能最优的引物为FS23,采用该引物对供试菌株的相应检测靶点进行PCR扩增,44株沙门氏菌都能扩增到一条492 bp特异性片段,而22株非沙门氏菌则不能扩增出这一特异性片段.以FS23为引物建立PCR方法检测猪霍乱沙门氏菌基因组DNA的灵敏度为11.9 fg/μL,细菌纯培养物灵敏度为4.9×102cfu/mL;用猪霍乱沙门氏菌人工污染牛奶样品,如果接种起始菌量为100 cfu/25 mL时,只需要增菌5 h,采用上述方法即能检测出沙门氏菌.[结论]引物FS23对应的基因序列是一个性能优良的新分子检测靶点,具备很高的特异性和灵敏性,能够广泛应用于食品中沙门氏菌的快速检测.     

Simultaneous detection of Fusarium asiaticum and Fusarium graminearum in wheat seeds using a real-time PCR method

Aims:  To develop a PCR-based method for quantitative detection of Fusarium asiaticum ( Fa ) and Fusarium graminearum ( Fg ) in wheat seeds.
Methods and Results:  Based on the sequences of the cyp51A gene, two primer pairs FaF + FaR and FgF + FgR were developed for the species-specific detection of Fa and Fg , respectively. To simultaneously detect these two phylogenetic species, a pair of primers FgaF + FgaR was developed based on the first and the second introns of β -tubulin gene. This primer pair amplified a 228-bp fragment only from Fa and Fg isolates, but not from 22 other Fusarium spp. and 13 other fungal species. A real-time PCR with this primer pair was able to quantify minute amounts of Fa and Fg DNA in wheat seeds rapidly.
Conclusions:  PCR primers designed based on the sequence of cyp51A or intron region of β -tubulin gene could allow differentiation of genetically related fungal species.
Significance and Impact of the Study:  The sensitive and quantitative detection method can be readily used in epidemiological studies and in assessing risk of Fusarium mycotoxin contamination in wheat samples.