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.

USE OF THE POLYMERASE CHAIN REACTION (PCR) TO DETECT MONOCLONALITY OF B CELL LYMPHO-PROLIFERATIVE DISORDERS

A technique has been developed using PCR to detect monoclonality of B-lymphoproliferative disorders. DNA was extracted from the blood, tissue and paraffin embedded sections by biochemical means or boiling. Forty cases of B-non Hodgkin's lymphoma (NHL), 15 cases of T-NHL, 8 cases of chronic lymphocytic leukemia, 17 cases of reactive lymphadenopathy and 12 cases of various non-lym-phocytic tumor were examined. Monoclonality of B-lymphocytes was detected in 86-92% of cases with B-lymphoproliferative diseases, but none in T-NHL, reactive disorders and non-lymphatic tumors. This technique provides a new molecular biologic method to diagnose malignant B-lymphoproliferative dicor-ders. It may be useful in Ig gene rearrangement study, differential diagnosis and retrospective investigation of lymphoproliferative disorders.

Comparison of direct fecal smear microscopy,culture,and polymerase chain reaction for the detection of Blastocystis sp.in human stool samples

Objective:To compare the sensitivity and specificity of direct fecal smear microscopy,culture,and polymerase chain reaction in the detection of Blastocystis sp.in human stool.Methods:Human stool samples were collected from a community in San Isidro,Rodriguez,Rizal,Philippines.These samples were subjected to direct fecal smear microscopy,culture and polymerase chain reaction to detect the presence of Blastocystis sp.Results:Of the 110 stool samples collected,28(25%)were detected positive for the presence of Blastocystis sp.by two or more tests.Culture method detected the highest number of Blastocystis-positive stool samples(n=36),followed by PCR of DNA extracted from culture(n=26),PCR of DNA extracted from stool(n=10),and direct fecal smear(n=9).Compared to culture,the sensitivity of the other detection methods were 66.7%for PCR from culture and 19.4%for both PCR from stool and direct fecal smear.Specificity of the methods was high,with PCR from culture and direct fecal smear having97.3%,while PCR from stool at 95.9%.Conclusions:In this study,in vitro culture is the best method for detecting Blastocystis sp.in human stool samples.

A quantitative polymerase chain reaction assay for the enumeration of brown tide algae Aureococcus anophagefferens in coastal waters of Qinhuangdao

Aureococcus anophagefferens, a small pelagophyte algae, has caused brown tide blooms in coastal waters of Qinhuangdao in recent years, presenting significant negative impacts on the shellfish mariculture industry. Under standard light microscopy, it is visually indistinguishable from other small algae in field samples due to its extremely small size. In this study, quantitative polymerase chain reaction（q PCR） based on 18 S r DNA sequences was developed and used to detect and enumerate A. anophagefferens. A linear regression（R2 = 0.91） was generated based on cycle thresholds value（Ct） versus known concentrations of A. anophagefferens. Twenty-two field samples collected in coastal waters of Qinhuangdao were subjected to DNA extraction and then analyzed using q PCR. Results showed that A. anophagefferens had a wide distribution in coastal waters along Qinhuangdao. Elevated A. anophagefferens abundance, category 3 brown tide blooms（〉200 000 cells/m L） occurred at Dongshan Beach and Tiger-stone Beach in August in 2013. In shellfish mariculture areas along coastal waters of Qinhuangdao, 4 stations had category 3 blooms, and 6 stations had category 2 blooms（35 000–200 000 cells/m L） in August and all stations had category 1 blooms（〉0 to ≤35 000 cells/m L） in October. Quantitative PCR allows for detection of A. anophagefferens cells at low levels in filed samples, which is essential to effective management and prediction of brown tide blooms.

BORRELIA BURGDORFERI DNA IN BIOLOGICAL SAMPLES FROM PATIENTS WITH SARCOIDOSIS USING THE POLYMERASE CHAIN REACTION TECHNIQUE

Polymerase chain reaction (PCR) was used to detect the presence of Barrelia burgdoferi DNA in biological samples from patients with sarcoidosis. The target DNA sequence was of chromosomal origin. The amplified DNA sequence was analyzed by agarose gel electrophoresis, PAGE with silver staining, and the identity of amplified DNA was confirmed by restriction enzyme cleavage and DNA-DNA hybridization with a 32P-labelled probe. The assay was sensitive to fewer than two copies of B. burgdorferi genome, even in the presence of a 104-fold excess of human eukaryotic DNA , and was also specific to different B. burgdorferi strains tested. Sera serologically positive to B. burgdcrferi (n=26), bronchoalveolar lavage fluid and supernatant of BALF (n=26) and peripheral blood (n=9) from sarcoidosis patients were tested. The positive rate was low (4/26, 2/26. and 0/9, respectively). It was considered that DNA from B. burgdor ferimay be identified in a minority of patients with sarcoidosis, and it may play a pathogenetic role in such cases. More studies need to be done before advancing the hypothesis of an etiologic role of B. burgdorferi in sarcoidosis.

Quantum dots induce hot-start effects for Taq-based polymerase chain reaction

Decent hot-start effects were here reported in Taq DNA polymerase-based polymerase chain reaction (PCR) when water-soluble CdTe quantum dots (QDs) were employed. The hot-start effects were revealed by the higher amplicon yields and distinguished suppression of nonspecific amplification after pre-incubation of PCR mix with quantum dots between 30°C and 56°C. DNA targets were well amplified even after PCR mixture was pre-incubated 3 hr at 30°C or 1 hr at 50°C. Importantly, the effects of QDs nanoparticles could be reversed by increasing the polymerase concentration, suggesting that there was an interaction between QDs and Taq DNA polymerase. Moreover, control experiment indicated that hot-start effect is not primarily due to the reduced polymerase concentration resulted from the above interaction. This study provided another good start to investigate potential implications of quantum dots in key molecular biology techniques.

Primary application of a real-time quantitative polymerase chain reaction for the detection of human breast cancer related novel gene-Metadherin expression

Objective:The aim of this study was to detect the expression level of Metadherin (MTDH) in peripheral blood of the breast cancer patients by real-time fluorescence quantitative polymerase chain reaction (PCR),and to explore the relationship between expression of Metadherin gene in the patients peripheral blood and the clinic-pathological features in breast cancer. Methods:Real-time fluorescence quantitative polymerase chain reaction was employed to determine the expression level of Metadherin gene in 80 peripheral blood samples of breast cancer patients and healthy donors. Results:The expression of Metadherin gene in breast cancer patients peripheral blood were positive,in which 34 breast cancer patients were highly expressed,accounting for 55.7%,while the expression of Metadherin gene in normal females peripheral blood were negative,there was statistical significance (Ratio = 2.02±0.81,P < 0.05); Ratio of the Metadherin expression in breast cancer patients peripheral blood and the glyceraldehyde-3-phosphate dehydrogenase expression was 1.15 ± 0.36. REST software analysis showed that the expression of Metadherin gene was significantly up-regulated in breast cancer. Conclusion:The SYBR Green I quantitative real-time polymerase chain reaction method can successfully detect the expression level of Metadherin gene. Expression level of Metadherin gene in breast cancer patients peripheral blood is closely related to survival,and it maybe involved in the development of breast cancer and used as an indicator of prognosis.

Sensitivity assay of polymerase chain reaction for detection of Canine Parvo Virus infection in dogs

A polymerase chain reaction was performed using re-ported primers for detection of Canine Parvo virus (CPV) in the stool sample obtained from repository. The PCR primers were specific to VP1/VP2 gene of CPV. Sensi-tivity assay of PCR detection was performed by making dilutions of CPV positive DNA extracted from fecal sample, carrying out PCR for each dilution and visualiz-ing amplicons in ethidium bromide stained agarose gel under UV radiation. Study was valuable in determining the efficiency of PCR. The sensitivity of PCR in present study was determined to be equivalent to detection of .00 2pg/μl of CPV DNA. The study was conducted to analyze the variation, sensitivity and repeatability.

Detection of Human Papillomavirus DNA in Oral Cancer Tissue Using Polymerase Chain Reaction

Using polymerase chain reaction (PCR), 25 specimens from 22 patients with oral carcinomas were examined by 6 selected primers of human papillomavirus (HPV). Eighteen of the 22 patients (18/22) gave positive reaction with a positive rate of 81.8%. The positive rates of HPV 6, 11, 16 and 18 were 27.3%, 18.2%, 63.6% and 40.9% respectively. 13.6% positive for mixed infection of HPV 16 and 18 (3/22) and 18.2% positive for mixed infection of HPV, 6, 11, 16 and 18 (4/22). Examining enlarged cervical lymph nodes in three cases with suspecting metastases to cervical lymph nodes from oral carcinomas. It revealed HPV DNA 16 and 18 in two cases and HPV DNA 18 in one case. These results suggested that there was a tendency for HPV 16 and 18 to metastasinze via lymphatics. Only one case of the three had a pathologic diagnosis of lymph node metastasis. Of the 30 non tumor controls, HPV DNA positivity was 10%, all being HPV 18. χ 2 test gave a P<0.005. It strongly indicated that HPV 16 and 18 were related to oral carcinomas.

Investigation on detection of Haemophilus ducreyi by Polymerase Chain Reaction

Objective:To investigate the application of polymerase chain reaction (PCR) detection of Haemophilus ducreyi in clinical diagnosis of chancroid. Methods: Nucleotide sequences of 16srRNA gene specific for H. dureyi were used to develop primer sets for amplification of two strains. The amplified products were tested via PCR and sequenced by electrophoresis in a 1.5% gel.These products were compared with those of heterogeneous species or related bacteria to test the specificity of the PCR assay. PCR amplification with different concentrations of H.ducreyi was performed to test its sensitivity. Results: PCR amplification of two strains of H. ducreyi produced a single band of expected 438bp length. The sequence was identified with genomic DNA. None of the other 19 reference species amplified under the same conditions gave this result. The highest sensitivity of PCR assay in the present test was 10ng/L. Conclusions: PCR assay for detection of H. ducreyi is a rapid, specific, and sensitive detection method. If laboratory conditions are strictly controlled, PCR assay is a potentially useful laboratory test for H. ducreyi infection diagnosis.

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%.

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.

基于主成分分析的多重定量PCR荧光串扰校正

聚合酶链式反应(PCR)是分子生物学常用的检测手段,主要用于对生物的DNA或RNA进行检测。由于荧光光谱重叠和滤光片过滤带宽限制,检测时所获得的荧光数据通常会包含荧光通道之间的串扰,串扰的存在使PCR结果分析变得复杂,并可能影响最终的检测结果。选择合适的光学元件,并确定通道间的补偿矩阵,可以降低甚至消除荧光串扰。目前荧光补偿矩阵大多通过迭代计算获得,还没有一种简单的方法可以从混合的多通道荧光数据中找到荧光补偿矩阵。为了快速获得荧光补偿矩阵,减小计算量,采用主成分分析法(PCA)中确定主成分的方式,基于搭建的测试平台进行单一染料实验,获得染料的荧光信号在各个检测通道的分布情况,计算得到荧光补偿矩阵。通过分析补偿矩阵,发现对于搭建的硬件系统,Cy5染料对Cy5.5通道串扰较大,串扰比例为8.76%,同时Cy5.5染料对Cy5通道串扰影响也相对较大,比例约为6.2%;其次是ROX染料对HEX通道串扰,比例约为2.68%;HEX染料对FAM通道串扰,比例约为1.58%;FAM染料对HEX通道串扰相对较小,比例约为0.25%,其余通道无明显串扰,与荧光光谱反映的结果一致。采用得到的荧光补偿矩阵对单一染料实验得到的原始荧光数据进行处理,有效去除了非目标通道的荧光串扰,实现了荧光通道数据的解耦,验证了方法的可行性。最后设计了染料颜色分辨实验,将不同浓度的多种染料进行组合测试,并采用所提出的方法将得到的数据进行荧光补偿。实验结果表明,荧光通道各自的线性相关性较高,五个荧光通道的线性相关系数r均大于0.99,该结果进一步验证了该补偿方法的有效性。

位点特异性PCR鉴别水牛角及其伪品

目的:开发一种针对水牛角的特异性聚合酶链式反应(PCR)鉴别方法,快速、准确地鉴别水牛角与其他混伪品。方法:通过比对水牛、黄牛、牦牛、山羊等动物的细胞色素C氧化酶亚基Ⅰ(COⅠ)基因序列差异,筛选水牛的特异性单核苷酸多态性(SNP)位点,设计出相应的鉴别引物,并对影响PCR体系的相关条件进行优化,考察验证方法的耐受性和适用性。结果:PCR条件为扩增体系20μL、退火温度60℃、循环次数32次、DNA模板量为100 ng、10μmol·L^(-1)正反引物对各0.8μL时,使用设计的鉴别引物SN1对水牛角进行PCR扩增,产物经琼脂糖凝胶电泳后出现约358 bp的特异性条带,其他物种的样品均无条带出现。结论:该PCR鉴别方法特异性好,能迅速、准确地鉴别水牛角和其他常见动物角类样品,为水牛角的真伪鉴别提供参考。

基于PCR-LFIA的非洲猪瘟病毒核酸检测方法研究

该文利用聚合酶链式反应(PCR)技术进行扩增后,以侧流免疫层析方法(LFIA)对核酸进行检测,建立了一种非洲猪瘟病毒核酸的快速检测方法。通过设计引物,构建了pUC57-ASFV-(1-1941)质粒作为阳性质控品,采用柠檬酸三钠还原法制备胶体金颗粒用于标记抗体建立免疫分析方法。通过优化实验条件,在质粒浓度1.6×10^(-2)~1.6×10^(8)copies/μL范围内,得到pUC57-ASFV-(1-1941)阳性质粒的检出限为1.6 copies/μL。该方法与其他猪病毒无交叉,特异性良好,可作为非洲猪瘟现场筛查的辅助方法。

基于嵌入式ARM的微流控PCR检测系统设计

为了实现微流控设备功能的集成化、提高交互系统的可操作性,提出一种基于嵌入式ARM的微流控聚合酶链式反应(PCR)控制系统及上位机软件的设计方案。系统硬件采用多个嵌入式ARM控制器与功能模块相互协调,制定可扩展的通信机制和协议,实现微流控PCR检测过程中的流路运动、温度控制、荧光信号采集等功能的集成控制。采用Qt结合SQLite数据库设计了上位机软件,利用多线程和节点映射,实现对硬件模块的协调控制以及检测信息存储。实验表明:该系统运行稳定,人机交互效果好,可操作性高,满足微流控PCR的集成功能需求。

旋转式三温区微流控PCR扩增平台的研制

微流控芯片用于聚合酶链式反应(PCR)可提高检测效率和自动化程度,但目前把核酸提取、扩增、检测功能集成到一块芯片上仍比较困难,此外,微流控芯片专用PCR仪也存在温度控制不够快速、精准的问题。作者采用空间上温度循环代替时间上温度循环的设计思路,搭建了一套旋转式三温区微流控PCR扩增平台,对大肠杆菌进行核酸提取和扩增。结果表明,旋转式三温区微流控PCR扩增平台拥有较好的热均匀性和热稳定性,平台的升降温速率分别为3.5℃/s和2.67℃/s,单次循环时间为110 s。与9700型PCR仪相比,所建平台的温度控制方案更为简单、升降温速率更高、循环时间更短。本研究结果可为实现核酸提取、扩增一体化的微流控PCR设备的研发提供参考。

天麻PCR‑层析试纸条快速可视化检测方法的建立与评价

目的通过聚合酶链式反应(PCR)与层析试纸条结合的方法,实现天麻快速可视化真伪鉴别,并对其检测效果进行评价。方法采用一步法提取天麻及其伪品基因组DNA,应用NCBI数据库设计天麻特异性引物。采用DNA分子克隆技术制备天麻阳性质粒,作为天麻阳性对照品。建立PCR-层析试纸条法鉴定天麻真伪,摸索最优实验条件,并进行方法学评价。结果①样品DNA提取的纯度符合要求,最优的PCR反应引物浓度为1μmol/L,循环为29次。②分子克隆的天麻阳性质粒序列与天麻DNA分子标记特异性指纹区片段序列同源性为98%,可作为天麻PCR-层析试纸条法的阳性对照品。③PCR-层析试纸条法的方法学评价结果显示:天麻对照药材在试纸条上出现两个条带,伪品和阴性对照品出现1个条带,与琼脂糖凝胶电泳法结果一致,特异性良好;PCR-层析试纸条法较琼脂糖凝胶电泳法的灵敏度高100倍,天麻DNA浓度为10-1 mg/L时,试纸条仍有模糊条带;在第3、6、9、12个月采用PCR-层析试纸条法进行检测,检测结果与预期一致,稳定性良好;混合样品验证显示,PCR-层析试纸条法的最低检测限为10%,而琼脂糖凝胶电泳法的最低检测限为50%。④采用PCR-层析试纸条法对15份市售天麻样品进行检测,鉴别出3个伪品,与琼脂糖凝胶电泳法结果一致。结论所构建的PCR-层析试纸条检测方法特异性强、灵敏度高、操作简便快速,可在短时间内实现鉴定结果的可视化,检测结果准确、稳定,为道地药材天麻的真伪鉴别提供了新方法。

PCR可视化检测技术的开发及其在猪源性检测中的应用

该文以G-四链体和硫磺素T为报告体系,建立一种聚合酶链式反应(polymerase chain reaction,PCR)可视化检测方法,仅需普通基因扩增仪和蓝光灯,即可在反应后通过肉眼观测荧光亮度直接判读结果,无需开盖,避免PCR产物污染问题的发生。并以猪源性DNA为检测目标设计特异性引物,建立猪源性可视化检测方法,通过对市售的牛、羊、兔、鸡、鸭肉对比,特异性良好,检测限达到0.01 ng/μL,可在混合肉DNA样品中检出1%的猪肉,并且能成功应用到实际食品样本的检测中。反应时间可控制在1 h以内,相较常用的实时荧光PCR,该方法既缩短时间,又不需要使用更昂贵的荧光PCR仪,且无需荧光基团修饰的探针,适合在简单的条件下进行快速定性检测。

基于微流控技术的数字PCR的发展及其应用

作为一种新兴的扩增检测技术,基于微流控的数字聚合酶链式反应(PCR)有着高通量、高灵敏度及高耐受性等优势,因而得到了研究者的广泛关注,其相关技术也在不断的发展中。综述了基于微流控的数字PCR的研究进展,重点讨论了基于微流控的数字PCR的液滴打印技术、多层微流控芯片技术、微珠技术、聚二甲基硅氧烷(PDMS)技术等的不同实现方式。介绍了基于微流控的数字PCR在定量检测、精准分子诊断、肿瘤个性化诊断以及食品安全检测中的应用。最后,对基于微流控的数字PCR技术目前存在的不足和问题进行了阐述,并对其未来的研究方向和发展趋势进行了展望。