Sequencing of hepatitis C virus cDNA with polymerase chain reaction directed sequencing *

AIM To explore a rapid and easy sequencing method for hepatitis C virus (HCV) genome, and establish a new sequencing method in China. METHODS Polymerase Chain Reaction (PCR) was combined with DNA sequencing technique. PCR products were purified by agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), Polyethylene glycol (PEG) respectively. Then in the presence of a 5′ labeling PCR primer, purified PCR products were directly sequenced. By this method, HCV NS5b cDNA from two HCV infected individuals (HC 42 and HC 49) were sequenced.

Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用

Taq DNA聚合酶作为实时荧光定量聚合酶链式反应(qPCR)技术的核心组分,其性能优劣直接影响qPCR技术的进一步发展。然而,野生型Taq DNA聚合酶的耐抑制剂性能差、延伸性能不足。为获得具有高性能的Taq DNA聚合酶,采用基因工程技术将双链DNA结合蛋白Sso7d或Sto7d融合在野生型Taq DNA聚合酶的N端或C端,构建了4个均可溶表达的改造体,再经过耐受性测试筛选较优的改造体,结果显示:改造体Taq-Sto的耐受性最高,其热稳定性不受影响,且在1 s/kbp的延伸条件下能成功扩增靶标,表明Taq-Sto具有增强的延伸性能,在TaqMan探针法qPCR体系中对腐殖酸、单宁酸、全血等抑制剂同样表现出良好的耐受性。EMSA实验发现:Taq-Sto对DNA模板的结合亲和力有所提高,有利于增强Taq-Sto对模板的竞争力;将Taq-Sto应用于非洲猪瘟病毒(ASFV)的TaqMan探针法qPCR检测,与商品化试剂相比,Taq-Sto具有更低的ASFV检出限,且在体积分数为2%~6%的猪粪便样本或猪肉样本中的检测灵敏度分别为100.0%和85.4%,说明Taq-Sto在直扩qPCR检测领域更具有优势。

Detection of bacterial DNA from cholesterol gallstones by nested primers polymerase chain reaction

AIM To search for bacterial DNA sequences in cholesterol gallstones with negative bacterial culture. METHODS DNA was extracted from cholesterol gallstones in gallbladders and nested primers polymerase chain reaction (NP PCR) was used to amplify bacterial gene fragments for identifying the existence of bacteria. The samples of bacterial DNA extracted from potentially causative or unrelated living bacteria were amplified in vitro as the standard markers and comparative 16S ribosomal RNA sequence analysis was made for bacterial identification. RESULTS The gallbladder gallstones of 30 patients were analyzed and bacterial DNA was found in 26 patients. Among them, gallstones with cholesterol content between 30%-69% were seen in 5 (5/5) patients, 70%-90% in 11 (11/14) patients, and more than 90% in 10 (10/11) patients. There was no difference either in cholesterol and water content of gallstones or in harboring bacterial DNA of gallstones. E.coli related DNA fragments appeared in the stones of 8 (26 67%) patients; propionibacteria type DNA in 7 (23 33%); and harbored bacterial gene fragments in 2 patients, similar to Streptococcus pyogenes . A more heterogeneous sequence collection was found in 7 (23 33%) patients, which could belong to multiple bacterial infections. Two (6 67%) patients had bacterial DNA with low molecular weight which might be related to some unidentified bacteria. CONCLUSION Most cholesterol gallstones harbor bacterial DNA. It is important to determine whether these microorganisms are innocent bystanders or active participants in cholesterol gallstone formation.

核酸适配体筛选中单链DNA制备方法的研究进展

核酸适配体是人工合成的短链核酸,作为分子识别元件,能够与各类靶标物质高特异性、高亲和力的结合,分为单链DNA和RNA两种类型。其中单链DNA适配体由于其稳定性比RNA适配体更好而更受欢迎,因此得到广泛应用。核酸适配体筛选通常是通过配体指数富集系统进化技术(systematic evolution of ligands by exponential enrichment,SELEX)实现的,筛选能否成功在很大程度上取决于其最关键的单链制备步骤,即将双链DNA转化为相应的单链DNA。目前,存在许多方法可以制备单链DNA,包括热变性法、生物素-链霉亲和素亲和分离法、变性胶电泳分离法、核酸外切酶消化法、不对称聚合酶链式反应(polymerase chain reaction,PCR)法等。本文在总结文献报道的基础上,具体阐述了各种单链DNA制备方法的原理、优缺点及近5年的应用情况,并对这些单链DNA制备方法进行了比较和展望,以期能为成功筛选各类靶标的核酸适配体提供参考。

Detection of hepatitis B viral DNA in liver with polymerase chain reaction

The sensitivity of PCR was determined for detection of HBV DNA in paraffin-embed-ded liver tissue with different methods for sample preparation.Of 8 cases of HBeAg-positiveHBV infection,HBV DNA was detected in 6 by extracting DNA from both frozen and dewaxedsamples,but in none by direct reaction.Of 12 cases subjected to Southern blot hybridization,HBV DNA was detected in 7 by this technique,in 10 by PCR with both methods of DNA extrac-tion and in 3 by direct PCR.The results showed that PCR was sensitive and was comparablewith blot hybridization in detecting intrahepatic HBV DNA.In comparison between differentmethods of sample preparation,the viral detection rate from the dewaxed samples was near thatfrom the frozen ones,while by the direct reaction HBV DNA could be detected only in a fewsamples with high level of infection.

A Molecular Approach to Identification of the Chinese Drug“pu Gong Ying”(Herba Taraxaci)and Six Adulterants byDNA Fingerprinting Using Random Primed PolymeraseChain Resaction(PCR)

DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbitrarily primed polymerase chain reaction(AP-PCR)and random amplified polymorphic DNA(RAPD).Distinctive,reproducible genomic fingerprints from DNA from 7 species belonged to Compositae were generated with two long(20 and 24 mer)and one short(10 mer)randomly chosen primers.The Pu Gong Ying can be differentiated from six species of Tu Gong Ying according to the banding pattems of their amplified DNA on agarose gels.The results showed that AP-PCR and RAPD methods can be used for identifying Chinese drugs.Moreover,the Similarity Indexes of the genomic DNA fingerprints showed that Pu Gong Ying and its adulterants are unrelated.Therefore,AP-PCR and RAPD methods can be used for identifying Chinese drugs.

SELEX技术中制备单链DNA的方法

制备单链DNA(ssDNA)是许多常用实验中的重要内容,是通过指数富集的配体系统(SELEX)进行体外筛选核酸适配体的关键步骤之一。目前已报道多种方式可通过双链DNA(dsDNA)制备ssDNA,包括链霉亲和素包被磁珠分离、不对称PCR、不等大小引物PCR、酶消化、不对称PCR结合酶消化和不对称乳液PCR等,而如何快速高效地制备ssDNA是研究重点。本文综述了常用的几种通过聚合酶链式反应(PCR)制备ssDNA的方法,对其进行综合分析。根据产物的纯度、操作难易、实验成本等方面综合考虑,选择产物高纯度、操作简便且实验成本较低的ssDNA制备方法,为具有不同需求的应用场景提供参考依据。

FQ-PCR检测乙型肝炎病毒DNA与时间分辨免疫荧光法检测乙肝五项在乙肝诊断中的应用价值

目的探讨荧光定量聚合酶链反应(FQ-PCR)检测乙型肝炎病毒(HBV)-DNA与时间分辨免疫荧光法检测乙肝五项在乙肝诊断中的应用价值。方法选择2021年1月至2023年10月本院接收的82例疑似乙肝患者作为研究对象,均通过FQ-PCR实施HBV-DNA检测,同时予以时间分辨免疫荧光法检测乙肝五项[乙肝病毒核心抗体(HBcAb)、乙肝病毒e抗体(HBeAb)、乙肝病毒e抗原(HBeAg)、乙肝表面抗原(HBsAg)、乙肝表面抗体(HBsAb)],并分析检测结果。结果82例患者中,HBV-DNA阳性率为70.73%(58/82)。82例患者中,组合A(HBcAb+HBeAg+HBsAg阳性)占比91.46%(75/82),组合B(HBeAb+HBsAg阳性)占比63.41%(52/82),组合C(HBcAb+HBeAb+HBsAg阳性)占比37.80%(31/82),组合D(HBcAb+HBeAb+HBsAb阳性)占比6.10%(5/82),组合E(HBeAg+HBsAg阳性)占比64.63%(53/82)。组合A、组合B、组合C、组合D、组合E中HBV-DNA阳性率分别为53.33%(40/75)、7.69%(4/52)、29.03%(9/31)、40.00%(2/5)、5.66%(3/53),组合A中HBV-DNA阳性率均高于其他各项组合,差异具有统计学意义(P<0.05)。FQ-PCR检测HBV-DNA联合时间分辨免疫荧光法检测乙肝五项诊断乙肝的准确度、灵敏度高于单独FQ-PCR检测HBV-DNA及时间分辨免疫荧光法检测乙肝五项,差异具有统计学意义(P<0.05)。结论在乙肝诊断中,FQ-PCR检测HBV-DNA与时间分辨免疫荧光法检测乙肝五项各具优势,积极联合应用有助于进一步提升诊断效能。

Pfu-sso7d DNA聚合酶的制备及反应缓冲液的研究

目的:制备有高效扩增长片段以及复杂片段能力的Pfu DNA聚合酶。方法:运用基因工程学方法构建重组质粒pET-28a-Pfu-sso7d,采用优化后的诱导条件对pET-28a-Pfu-sso7d实行诱导表达,将菌液超声破碎并提取上清液,应用镍亲和层析洗脱纯化取得较纯Pfu-sso7d DNA聚合酶。采用qPCR方法对酶进行酶活力单位定量、确定反应缓冲液组分最佳浓度及检测自制Pfu-sso7d DNA聚合酶酶活性。结果:在1 mmol·L^(-1) IPTG、16℃条件下诱导Pfu-sso7d DNA聚合酶菌液16 h,Pfu-sso7d DNA聚合酶高效表达;用20~100 mmol·L^(-1)咪唑可将蛋白洗脱。Pfu-sso7d DNA聚合酶最终活力单位定量为1 U·μL^(-1)。PCR最佳反应缓冲液为pH9.0、20 mmol·L^(-1) Tris-HCl、0.6 mmol·L^(-1) MgSO_(4)、50 mmol·L^(-1) KCl、5 mmol·L^(-1)(NH_(4))_(2)SO_(4)、0.1%Triton X-100,添加剂组分为3%DMSO、1 mol·L^(-1)甜菜碱。Pfu-sso7d DNA聚合酶能高效扩增3000 bp的目的条带,活性达到商用高保真DNA聚合酶水平。结论:基于基因重组制备的Pfu-sso7d DNA聚合酶可进行高效的PCR反应,节省了实验室PCR成本,为进一步提高Pfu DNA聚合酶活性提供一定技术参考。

Small Amplicons Mutation Library for Vaccine Screening by Error-Prone Polymerase Chain Reaction

Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.

Development of a droplet digital polymerase chain reaction assay for the sensitive detection of total and integrated HIV-1 DNA

Background:Total human immunodeficiency virus(HIV)DNA and integrated HIV DNA are widely used markers of HIV persistence.Droplet digital polymerase chain reaction(ddPCR)can be used for absolute quantification without needing a standard curve.Here,we developed duplex ddPCR assays to detect and quantify total HIV DNA and integrated HIV DNA.Methods:The limit of detection,dynamic ranges,sensitivity,and reproducibility were evaluated by plasmid constructs containing both the HIV long terminal repeat(LTR)and human CD3 gene(for total HIV DNA)and ACH-2 cells(for integrated HIV DNA).Forty-two cases on stable suppressive antiretroviral therapy(ART)were assayed in total HIV DNA and integrated HIV DNA.Correlation coefficient analysis was performed on the data related to DNA copies and cluster of differentiation 4 positive(CD4^(+))T-cell counts,CD8^(+)T-cell counts and CD4/CD8 T-cell ratio,respectively.The assay linear dynamic range and lower limit of detection(LLOD)were also assessed.Results:The assay could detect the presence of HIV-1 copies 100%at concentrations of 6.3 copies/reaction,and the estimated LLOD of the ddPCR assay was 4.4 HIV DNA copies/reaction(95%confidence intervals[CI]:3.6-6.5 copies/reaction)with linearity over a 5-log_(10)-unit range in total HIV DNA assay.For the integrated HIV DNA assay,the LLOD was 8.0 copies/reaction(95%CI:5.8-16.6 copies/reaction)with linearity over a 3-log 10-unit range.Total HIV DNA in CD4^(+)T cells was positively associated with integrated HIV DNA(r=0.76,P<0.0001).Meanwhile,both total HIV DNA and integrated HIV DNA in CD4^(+)T cells were inversely correlated with the ratio of CD4/CD8 but positively correlated with the CD8^(+)T-cell counts.Conclusions:This ddPCR assay can quantify total HIV DNA and integrated HIV DNA efficiently with robustness and sensitivity.It can be readily adapted for measuring HIV DNA with non-B clades,and it could be beneficial for testing in clinical trials.

荧光定量PCR检测尿沉渣TB-DNA在泌尿系结核病诊断中的应用

目的探讨荧光定量聚合酶链反应(FQ-PCR)检测尿沉渣结核病-DNA(TB-DNA)在泌尿系结核病(UTB)诊断中的应用。方法回顾性分析南阳市中心医院2021年4月至2022年4月收治的疑似为UTB的61例患者尿液沉渣标本实验室系统检测资料,以上患者就诊期间均已完成FQ-PCR检测法、结核分枝杆菌(MTB)涂片检测与MTB培养法,最终确诊为UTB31例、非UTB30例,比较3种检验方法诊断UTB的效能。结果FQ-PCR检测尿沉渣TB-DNA敏感度、特异度、准确率和阳性率明显高于MTB涂片检测,差异有统计学意义(P<0.05);FQ-PCR检测敏感度、准确率、阳性率明显高于MTB培养法,差异有统计学意义(P<0.05),但特异度与其无明显区别,差异无统计学意义(P>0.05)。结论FQ-PCR检测尿沉渣TB-DNA用于UTB诊断具有较高的敏感度、特异度与准确率,可减少早期UTB诊断漏诊率。

Development and Clinical Application of a Single-tube Nested PCR Method to Amplify the DNA Polymerase Ⅰ Gene of Treponema Pallidum

Objective: To develop a sensitive, specific and simple method for detection of extremely low numbers of T. pallidum in clinical specimens, as a significant addition to the serologic tests for syphilis diagnosis. Methods: Double-tube nested PCR(DN-PCR) and single-tube nested PCR(SN-PCR) assays were performed to amplify specific fragments of the DNA poly-merase I gene(polA) of T. pallidum. Sensitivity and specificity of the two PCR assays were tested. Eighty-six whole blood specimens from persons with suspected syphilis were detected by the two nested PCR methods. The TPPA test was used as a comparison for detecting syphilis in sera from corresponding patients. Results: Only specific amplicons could be obtained during amplification of the T. pallidum polA gene and the detection limit was approximately 1 organism when analyzed on gel by the two PCR methods. Of 86 clinical specimens, 62 were positive by TPPA. Of these, 54 and 51 were positive by the DN-PCR and SN-PCR, respectively, which does not represent a statistically significant difference between the two PCR tests. Of 24 TPPA-negative specimens, 5 were positive by both DN-PCR assay and SN-PCR assay. Conclusion: The SN- polA PCR method is extremely sensitive, specific and easy to perform for detecting low numbers of T. pallidum in clinical blood specimens as a complementary to serology for syphilis diagnosis.

Optimization of PCR Reaction System for Random Single-strand DNA Pool in SELEX Technology

[Objective] This study aimed to optimize the PCR amplification conditions for random ssDNA pool in SELEX technology. [Method] L16（45） orthogonal experimental design was adopted for optimization of five important factors affecting PCR reaction system for random single-stranded DNA pool including Mg2＋ concentration, dNTP concentration, amount of Taq DNA polymerase, primer concentration and amount of random single-stranded DNA pool at four levels. Meanwhile, the annealing temperature and number of PCR reaction cycles were optimized to establish the optimal reaction system and PCR procedure. [Result] The optimal combination of PCR reaction system for random ssDNA pool was obtained, with a total system volume of 20 μl containing 2.0 μl of 10 × Buffer, 0.5 ng of random ssDNA pool, 2.5 mmol/L Mg2＋, 0.25 mmol/L dNTP Mixture, 0.6 μmol/L upstream and downstream primers and 1.5 U of Taq DNA polymerase; the optimal annealing temperature was 68 ℃ and the optimal number of cycles was 12. Under the above conditions, clear and stable bands with high specificity for random ssDNA pool were amplified. [Conclusion] This study laid the foundation for selection of parameters with higher specificity in SELEX technology.

基于DNA条形码和特异性PCR技术鉴别蚂蟥及其常见伪品

目的:建立蚂蟥特异性PCR鉴别方法,能够快速准确地鉴别蚂蟥与其常见伪品。方法:通过分析蚂蟥与其常见伪品的细胞色素C氧化酶亚基Ⅰ(COⅠ)序列,寻找蚂蟥SNP变异位点,设计特异性引物;通过对退火温度、循环次数、DNA模板量及Taq酶等关键因素的考察,确立最优反应体系及条件,并将特异性PCR方法应用到水蛭(蚂蟥)粉末中进行药材粉末的基原鉴别,同时为保证试验结果的准确性,将PCR扩增产物进行一代测序。结果:特异性PCR结果表明蚂蟥在400~600 bp有单一明亮条带,常见混伪品则无此条带,试验结果与一代测序结果一致,为蚂蟥。结论:该方法能够将蚂蟥与其常见伪品菲牛蛭、东北小水蛭、黑条、小黑条等区别,且能鉴别水蛭(蚂蟥)粉的基原,为提升中药监管力度,打击中药掺伪行为提供了强有力的技术支持。

数字PCR技术检测肺腺癌循环肿瘤DNA中EGFR突变的临床意义

目的探讨数字聚合酶链反应(droplet digital polymerase chain reaction,ddPCR)技术检测肺腺癌循环肿瘤DNA(circulating tumor DNA,ctDNA)中表皮生长因子受体(epidermal growth factor receptor,EGFR)突变的临床意义。方法采用ddPCR技术检测肺腺癌患者102例的ctDNA中EGFR突变即EGFR-L858R突变和EGFR-19del突变。分析ctDNA与患者临床参数的关系。绘制ROS曲线,评估ctDNA对患者预后和是否发生转移的预测作用。结果ddPCR技术检测ctDNA中EGFR突变评价EGFR突变状态的曲线下面积(area under the curve,AUC)为0.694,敏感度为40.2%,特异度为88.6%。与早期肺腺癌相比,晚期肺腺癌患者ctDNA中EGFR突变检测的AUC(0.837 vs.0.498)和敏感度(75.2%vs.10.2%)更高。肿瘤发生转移与ctDNA中EGFR突变丰度呈强的正相关性(r=0.510,P<0.001)。ctDNA阳性组的无进展生存期、总生存时间明显短于ctDNA阴性组(13.8个月vs.42.2个月,34.4个月vs.66.8个月,P<0.05),接受人表皮生长因子受体酪氨酸激酶抑制剂治疗的ctDNA阳性组无进展生存期短于ctDNA阴性组(12.0个月vs.24.0个月,P<0.05);此外,ctDNA阳性患者的总生存时间较阴性组缩短(34.4个月vs.66.8个月,P<0.05)。结论ddPCR技术可用于检测晚期肺腺癌患者中ctDNA中EGFR突变,ctDNA中EGFR阳性提示患者可能存在远处转移和预后差。

The cloning of 3'-truncated preS/S gene from HBV genomic DNA and its expression in transgenic mice

INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes

Sequencing of PCR amplified HBV DNA pre-c and c regions in 2.2.15 cells and antiviral action by targeted antisense oligonucleotide directed against sequence

AIM To study the specific inhibition of HBV gene expression by liver-targeting antisense oligonucleotide (ASON) directed against pre-c and c regious in a sequence-specific manner.METHODS According to the result of direct sequencing of PCR amplified products, a 16-mer phosphorothioate analogue of the antisense oligonucleotide (PS-ASOn) directed against the HBV U5-like region was synthesized and then linked with one live-targeting ligand, the galactosylated poly-L-lysine. Their effect on the expression of HBV gene was observed using the 2.2.15 cells.RESULTS HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw1 by sequencing so that antisense oligonucleotides could bind specifically to the target sequence through base piring. Under the same experimental conditions, the inhibitory rates of PS-ASON to HBsAg and HBeAg were 70% and 58% at a concentration of 10μmol/L, while by ligand-PS-ASON they were 96% and 82%, the amount of HBV DNA in cultured supernatant and cells was reduced significantly. An unrelated sequence oligonucleotide showed no effectiveness. All the oligonucleotides had no cytotoxicity.CONCLUSION Antisense oligonucleotides complexed by the liver-targeting ligand can be targeted to cells via asialoglycoprotein receptors, resulting in supecific inhibition of HBV gene expression and replication.

Cloning and expression of NS3 cDNA fragment of HCV genome of Hebei isolate in E.coli

AIM To obtain greater antigenicity of HCV NS3 protein. METHODS The HCV NS3 cDNA fragment was amplified by reverse transcription polymerase chain reaction from the sera of the HCV infected patients. The DNA sequence was determined by dideoxy mediated chain termination method using T7 polymerase. HCV NS3 protein was expressed in E. coli . RESULTS Sequence analysis indicated that the HCV isolate of this study belongs to HCV Ⅱ; SDS PAGE demonstrated an M r 23800 and an M r 22000 recombinant protein band which amount to 14% and 11% of the total bacterial proteins separately. Western blotting and ELISA showed NS3 protein possessed greater antigenicity. CONCLUSION Recombinant HCV NS3 protein was expressed successfully, which provided the basis for developing HCV diagnostic reagents.

Rapid detection of sepsis complicating acute necrotizing pancreatitis using polymerase chain reaction

INTRODUCTIONAcute narcotizing pancreatitis usually takes a severe clinical course and is associated with multiple organ dysfunction .With the further understanding of pathophysiological events of acute pancreatisis and the therapeutic measuses taken by the clinicians ,the patients can pass through the critical carry stages ,and then the septic complication caused by rtanslocated bacteria, mostly gram-negative microbes from the intestines ensues[1].