肠出血性大肠杆菌(O157：H7)的特异性荧光探针检测方法的建立

目的:建立一种real-time PCR,快速准确检测肠出血性大肠杆菌O157:H7。方法:以肠出血性大肠杆菌0157:H7 rfbE为待检靶基因,设计一对引物和一条Taqman探针,探针5’端用FAM基团标记,3’端用TAMRA标记。通过重组质粒的构建,建立并优化了大肠杆菌0157:H7的荧光定量PCR检测方法。结果:在人工污染样本无需富集的情况下,检测的最低DNA浓度是10拷贝/反应(3CFU/mL);特异性检测实验中,0157菌株检测结果均为rfbE阳性,而非0157:H7菌株检测结果均为阴性;重复性实验中,批内、批间变异系数均小于3%。结论:实验结果显示此荧光定量PCR方法特异性、灵敏度高,重复性好,可对分离的可疑大肠杆菌0157:H7菌株进行快速鉴定。     

Quantitation of the residual DNA from rice-derived recombinant human serum albumin

Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2 × 10⁴ to 0.2 pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8 ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.     

Quantitative detection of residual E. coli host cell DNA by real-time PCR

E. coli has been widely used as a host system to manufacture recombinant proteins for human therapeutic use. Among impurities to be eliminated during the downstream process, residual host cell DNA is a major interest for safety. Residual E. coli host cell DNA in the final products are usually determined using conventional slot blot hybridization assay or total DNA Threshold assay, although these methods are time consuming, expensive, and relatively insensitive. Therefore a sensitive real-time PCR assay for specific detection of residual E. coli DNA was developed and compared with slot blot hybridization assay and Threshold assay to validate the overall capability of these methods. Specific primer pair for amplification of the E. coli 16S rRNA gene was selected to improve the sensitivity, and E. coli host cell DNA was quantified by use of SYBR Green 1. The detection limit of real-time PCR assay in the optimized condition was calculated to be 0.042 pg genomic DNA, which is much higher than those of slot blot hybridization assay and Threshold assay of which detection limit were 2.42 and 3.73 pg genomic DNA, respectively. The real-time PCR assay was validated to be more reproducible, accurate, and precise than slot blot hybridization assay and Threshold assay. The real-time PCR assay may be a useful tool for quantitative detection and clearance validation of residual E. coli DNA during the manufacturing process for recombinant therapeutics.     

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).     

Development of a real time polymerase chain reaction for quantitation of Schistosoma mansoni DNA

This report describes the development of a SYBR Green I based real time polymerase chain reaction (PCR) protocol for detection on the ABI Prism 7000 instrument. Primers targeting the gene encoding the SSU rRNA were designed to amplify with high specificity DNA from Schistosoma mansoni, in a real time quantitative PCR system. The limit of detection of parasite DNA for the system was 10 fg of purified genomic DNA, that means less than the equivalent to one parasite cell (genome ~580 fg DNA). The efficiency was 0.99 and the correlation coefficient (R(2)) was 0.97. When different copy numbers of the target amplicon were used as standards, the assay could detect at least 10 copies of the specific target. The primers used were designed to amplify a 106 bp DNA fragment (Tm 83 degrees C). The assay was highly specific for S. mansoni, and did not recognize DNA from closely related non-schistosome trematodes. The real time PCR allowed for accurate quantification of S. mansoni DNA and no time-consuming post-PCR detection of amplification products by gel electrophoresis was required. The assay is potentially able to quantify S. mansoni DNA (and indirectly parasite burden) in a number of samples, such as snail tissue, serum and feces from patients, and cercaria infested water. Thus, these PCR protocols have potential to be used as tools for monitoring of schistosome transmission and quantitative diagnosis of human infection.     

采用分子生物学技术诊断隐球菌脑膜脑炎的研究

目的采用分子检测技术对疑似隐球菌感染的脑膜炎病例进行诊断。方法收集患者的脑脊液样本,提取DNA,设计引物进行PCR扩增,采用DNA芯片技术对扩增产物进行分子检测。结果显示样本新生隐球菌阳性。结论通过ITS保守序列设计引物进行PCR扩增和DNA芯片技术对常规真菌学检查不能确定的疑似隐球菌脑膜炎患者脑脊液样本进行非培养检测,具有实验室诊断参考价值。     

Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays

Aims: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. Methods and Results: TaqMan^® assays were designed to target the IS900 and f57 genetic elements of Map. Both real‐time PCR assays were integrated with the Adiapure^® Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml^?1, 2·8 CFU g^?1 and 30 CFU g^?1 for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD’s for the f57 assay were 6·2 CFU ml^?1, 26·7 CFU g^?1 and 316 CFU g^?1. Conclusion: The integrated Adiapure^® extraction – IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map‐DNA in cheese and whole milk powder. Significance and Impact of the Study: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.     

Microarrays for the detection of HBV and HDV

The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.     

Development of a multiplex polymerase chain reaction for the simultaneous detection of microsporidians, nucleopolyhedrovirus, and densovirus infecting silkworms

We have developed a novel PCR-based assay for individual and simultaneous detection of three major pathogens (microsporidians, nucleopolyhedrovirus (NPV) and densovirus (DNV)) infecting the silkworm, Bombyx mori. Multiplex PCR, using three primer pairs, two of which were designed from the conserved regions of 16S small subunit ribosomal RNA gene of microsporidians, and polyhedrin gene of NPVs respectively, and a third primer pair designed from the internal sequences of B. mori DNVs (BmDNV), showed discrete and pathogen specific PCR products. The assay showed high specificity and sensitivity for the pathogenic DNA. Under optimized PCR conditions, the assay yielded a 794 bp DNA fragment from Nosema bombycis, 471 bp fragment from B. mori NPV (BmNPV) and 391 bp fragment from BmDNV. Further, this detection method was successfully applied to other silkworm species such as Antheraea mylitta and Samia cynthia ricini, in detecting same or similar pathogens infecting them. This method is a valuable supplement to the conventional microscopic diagnostic methods and can be used for the early detection of pathogens infecting silkworms. Furthermore it can assist research and extension centers for the safe supply of disease-free silkworms to farmers.     

Development of a real‐time TaqMan PCR assay for the detection of porcine and bovine Torque teno virus

Aims: The goal of this study was to develop and to optimize molecular tools to detect the presence of Torque teno virus (TTV) in swine and cattle. A novel real‐time polymerase chain reaction (PCR) using a TaqMan probe was developed to detect both genogroups of TTV strains. Methods and Results: Oligonucleotide primers and hybridization probes were designed based on sequence analysis of the noncoding region, a highly conserved part of the genome. The real‐time PCR assay specifically detected bovine and porcine TTV DNA without cross‐amplification of other common pathogens. The assay was compared with conventional PCR and nested‐PCR assays for the detection of porcine genogroups 1 and 2 and bovine TTV on plasma and faecal samples, and the assay was found faster, more reliable and reduced the risk of false positive results. Conclusions: The real‐time PCR assay provided better detection results for the two TTV genogroups in both swine and cattle compared to the conventional PCR assays. Significance and Impact of the Study: This new TaqMan PCR assay will be a useful tool for the detection of animal TTV strains, to evaluate the viral load from animal host and finally to identify the presence of these viruses in the agri‐food continuum.     

Multiplex PCR to detect four different tomato-infecting pathogens

This work was aimed to develop a multiplex PCR assay to detect infectious agents such as Clavibacter michiganensis subsp. michiganensis, Fusarium sp, Leveillula taurica, and begomoviruses in tomato (Solanum lycopersicum) plants. Specific primer sets of each pathogen were designed based on intergenic ribosomal RNA sequences for the first three, whereas for begomoviruses, primers were designed based on conserved regions. The design also considered that the length (200–800 bp) of the PCR products was resolvable by electrophoresis; thus 296, 380, 457, and 731 bp fragments for Clavibacter, Fusarium, Leveillula, and begomoviruses, respectively, were considered. PCR conditions were optimized to amplify all the products in a single tube from genomic DNA and circumvent PCR inhibitors from infected plants. Finally, when the multiplex PCR assay was tested with tomato plants infected with any of the four pathogens, specific PCR products confirmed the presence of the pathogens. Optimized PCR multiplex allowed for the accurate and simultaneous detection of Clavibacter, Fusarium, Leveillula, and begomoviruses in infected plants or seeds from tomato.     

A PCR-based approach to sequence the Candida tropicalis HSP90 gene

The heat shock protein 90 (hsp90) gene sequence is known to be highly conserved across the species barrier. A PCR-based method was thus utilised in an attempt to sequence the Candida tropicalis hsp90 gene. Primers for PCR were designed from conserved regions of the gene, which were identified by comparing the Saccharomyces cerevisiae and Candida albicans hsp90 gene sequences. Different sets of primers were designed to amplify and obtain overlapping DNA sequences of the C tropicalis gene. PCR was carried out on genomic DNA of Candidca tropicalis and the PCR products were cloned into suitable vector molecules for sequencing. In this way, a 2,070-basepair sequence of the C. tropicalis hsp90 gene was obtained. The PCR-based approach proved to be an easier method of obtain the sequence of a highly conserved gene, as compared to more conventional methods.     

Heavy metals contamination in shallow groundwater of a coal-mining district and a probabilistic assessment of its human health risk

To manage public health and make better use of groundwater resources, the concentration characteristics and a health risk assessment of eight heavy metals in shallow groundwater were studied. Besides this, this paper introduced triangular fuzzy numbers into the USEPA health risk assessment model to assess the health risk posed to local children and adults through different exposure pathways. The results showed that Mn levels exceeded the WHO’s guideline values of 100?µg/L with the proportion of 27.98% and Sr were over the health reference level (HRL) of 1500?µg/L with the proportion of 56.25%, while other heavy metals were below the corresponding standard. The results of the HRA showed that the non-carcinogenic risks from Sr and Mn in the district were relatively higher, while those from the remaining six heavy metals were relatively lower. All hazard index (HI) values did not exceed the safety level of 1 for either age group. The average carcinogenic risk from Cr was slightly higher than the acceptable level of 1?×?10^?6 for adults. Sensitivity analyses conducted using Monte Carlo simulation indicated that Sr and Cr concentrations were the most influential variables contributing to the non-carcinogenic and carcinogenic risk values, respectively, while body weight had a minor contribution.     

Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence

To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (<25%) of the GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.     

应用LNA-PCR法检测乙型肝炎病毒阿德福韦酯耐药位点基因突变

目的:建立简便、快速、灵敏的锁核酸(locked nucleic acid,LNA)探针实时荧光聚合酶链反应(PCR)检测方法,检测乙型肝炎病毒(hepatitis B virus,HBV)阿德福韦酯(Adefovir dipivoxil,ADV)耐药相关位点(rtA181V、rtN236T)突变。方法:通过基因测序筛选阳性样本,进而构建ADV rt181和rt236位点野生株和突变株重组质粒,设计包含扩增阿德福韦酯rtA181V和rtN236T耐药位点在内的特异性引物和LNA荧光探针,以构建的重组质粒为标准品建立实时荧光PCR反应体系,并通过与基因测序平行检测血清样本以判断检测方法的可行性与准确性。结果:所建立的LNA-PCR法能够检测102copies/ml的HBV中ADV基因突变,同时具备较高的特异性。通过对89例ADV治疗一年后HBV阳性临床样本进行检测,有8例(8.98%)rtA181V突变、5例(5.61%)rtN236T突变、2例(2.24%)rtA181V和rtN236T混合突变,检测结果与测序结果一致。结论:所建立的LNA-PCR法是一种简便、快速、灵敏的基因突变检测方法,能有效的区分单碱基突变,对慢性乙型肝炎患者德福韦治疗过程中耐药突变的监控和抗病毒药物的调整具有指导意义。     

基于TaqMan探针的蜜蜂囊状幼虫病病毒荧光PCR检测方法的建立和应用

为建立快速有效的蜜蜂囊状幼虫病检疫方法, 依据TaqMan荧光标记探针技术原理, 针对蜜蜂囊状幼虫病病毒保守序列, 设计出一对特异性引物和一条探针, 建立了一种快速检测蜜蜂囊状幼虫病病毒的荧光PCR方法。该方法对蜜蜂囊状幼虫病的检测具有较好的特异性, 与蜜蜂急性麻痹病病毒、蜜蜂慢性麻痹病病毒、蜜蜂残翼病病毒和黑蜂王台病病毒之间均无交叉反应。检测灵敏度可达1.0×10²拷贝/μL阳性质粒, 可对低病毒含量的样品进行准确检测。重复性和稳定性试验结果显示, 变异系数为1.6%, 说明该方法具有较好的重复性和稳定性。应用该方法对蜜蜂及蜂制品进行检测, 结果显示所建立的荧光PCR检测方法4 h内即可报告检测结果, 该方法具有快速、灵敏、特异及重复性好等优点, 适用于蜜蜂及其制品中蜜蜂囊状幼虫病病毒的快速检疫。     

Rapid simultaneous detection and identification of six species Candida using polymerase chain reaction and reverse line hybridization assay

The aim of this study was to develop and evaluate PCR based reverse line blot (RLB) hybridization assay for rapid detection of the most common Candida isolates from clinical specimens. A pair of universal primers targeting the ITS2 region of the gene from 28S rRNA to 5.8S rRNA was designed for PCR amplification of DNA from 6 Candida species (C. albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, C. dubliniensis), the reverse primer was biotin labeled. PCR products, which were 302-441 bp length, were hybridized with 6 specific oligonucleotides probes immobilized on a nylon membrane. These 6 probes proved specific (they hybridized with only their target molecules). The assay was shown to be sensitive in detecting yeast to a concentration of 10 CFU/ml. This method was used to test 100 isolates and 200 vaginal swabs. The results agreed with those of culture for all but 3 of 100 isolates. Sequencing was performed on these 3 samples and confirmed that the culture results were inaccurate. Our results show the PCR-RLB positive rate (49%) is higher than culture (39%) and smear microscopic screening (27%) (P<0.05). In conclusion, the PCR/RLB developed in this study is specific and offers increased sensitivity compared to culture for the detection of Candida species in swab specimens. Moreover, the improved detection of cases of polycandidal candidiasis is advantageous.     

荧光定量PCR检测重组新蛭素中毕赤酵母基因组DNA的残留量

目的:建立real-time PCR定量检测毕赤酵母基因组DNA残留量的方法,提高重组新蛭素的产品安全性。方法:选择拷贝数高且分布广泛的毕赤酵母5S rRNA基因为靶标基因设计扩增引物,提取酵母基因组DNA,稀释后作为扩增模板。以罗氏荧光定量PCR_LightCycler480平台为基础,建立基于SYBR GreenⅠ荧光染料的real-timePCR的检测方法,并考察用该方法检测重组新蛭素中毕赤酵母基因组残留量的灵敏度、精密度和回收率。结果:该法检测宿主DNA残留量灵敏度高,DNA浓度为0.1~1000 pg/μL范围内呈现良好的线性关系,其标准曲线的误差值小于0.2;用该法对5批注射用重组新蛭素(酵母)产品中宿主基因组DNA残留量进行了测定,结果分别为0.03、2.3、0.2、0.6、0.2 pg/mg。结论:该方法具有操作简便、灵敏度高等优点,可用于重组产品中酵母基因组残留DNA的定量测定。     

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。