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.

Detection of Human Parvovirus B19 Nonstrutural Protein DNA by Nested-Polymerase Chain Reaction in Gravida Serum and Pregnant Tissues

A new nested-polymerase chain reaction （nested-PCR） assay was developed to detect human parvovirus B19 DNA corresponding to the nonstructural protein in clinical specimens in a routine diagnostic laboratory. The sensitivity of this highly specific assay was up to 0. 005 fg of B19 DNA. Parvovirus B19 was identified in sera of 20 pregnant women with abnormal pregnant outcome. Among these 20 cases, intrauterine parvovirus infection did exist in 7 pregnant women because parvovirus B19 DNA was detected in the pregnant tissues of them such as placenta tissues, chorionic villi, amniotic fluid, fetal spleen, liver and abdominal fluids.

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。

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.

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。

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.

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。

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.

Detection of the Pandemic H1N1/2009 Influenza A Virus by a Highly Sensitive Quantitative Real-time Reverse-transcription Polymerase Chain Reaction Assay

A quantitative real time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers recommended by the World Health Organization (WHO) has been widely used successfully for detection and monitoring of the pandemic H1N1/2009 influenza A virus. In this study, we report the design and characterization of a novel set of primers to be used in a qRT-PCR assay for detecting the pandemic H1N1/2009 virus. The newly designed primers target three regions that are highly conserved among the hemagglutinin (HA) genes of the pandemic H1N1/2009 viruses and are different from those targeted by the WHO-recommended primers. The qRT-PCR assays with the newly designed primers are highly specific, and as specific as the WHO-recommended primers for detecting pandemic H1N1/2009 viruses and other influenza viruses including influenza B viruses and influenza A viruses of human, swine, and raccoon dog origin. Furthermore, the qRT-PCR assays with the newly designed primers appeared to be at least 10-fold more sensitive than those with the WHO-recommended primers as the detection limits of the assays with our primers and the WHO-recommended primers were 2.5 and 25 copies of target RNA per reaction, respectively. When tested with 83 clinical samples, 32 were detected to be positive using the qRT-PCR assays with our designed primers, while only 25 were positive by the assays with the WHO-recommended primers. These results suggest that the qRT-PCR system with the newly designed primers represent a highly sensitive assay for diagnosis of the pandemic H1N1/2009 virus infection.

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

ＢＯＲＲＥＬＩＡＢＵＲＧＤＯＲＦＥＲＩＤＮＡＩＮＢＩＯＬＯＧＩＣＡＬＳＡＭＰＬＥＳＦＲＯＭＰＡＴＩＥＮＴＳＷＩＴＨＳＡＲＣＯＩＤＯＳＩＳＵＳＩＮＧＴＨＥＰＯＬＹＭＥＲＡＳＥ ＣＨＡＩＮ ＲＥＡＣＴＩＯＮ ＴＥＣＨＮＩＱＵＥＬｉａｎＷｅｉ（连伟）ａｎｄ...

Detection and amplification of Helicobacter pylori urease gene A in gastric biopsies by using nested polymerase (?)hain reaction

A nested polymerase chain reaction(N-PCR)for the spegific detection of Helicobacterpylori(H.pylori)was developed with two primer pairs(nested primers)derived from ureasegene A of H.pylori.The N-PCR was used to detect 21 different samples of H.pylori including20 clinical isolates and 1 reference strain NCTC 14126,but it was negative for other bacterialspecies,showing the N-PCR assay to be 100% specific.Tenfold serial dilution experiments re-vealed the detection of as little as 0.1 fg of H.pylori DNA by N-PCR.To evaluate the PCR as-say for clinical samples,gastric biopsies were tested with N-PCR,and the results were comparedwith those of culture,urease test and histologic examination(reference standard,RS).In 30biopsy specimens,H.pylori DNA sequences were detected by PCR in all of 20(100%)positivetissue and none of the 10 negative tissues.PCR is a specific and sensitive method that can detectthe presence of H.pylori without the need for culture and would have significant importance di-agnostically and epidemiologically.

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.

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。

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%.

进口锦鲤暴发病病原的nested-PCR鉴定

为了查明锦鲤暴发性疾病的病因 ,将患病锦鲤的脑、肝、脾和肾等组织悬液接种到CO、CK和EPC等鱼类细胞系中培养 ,均未发现细胞病变 ;从病灶中取样进行病原菌的分离和培养 ,证明锦鲤有副溶血弧菌和嗜水气单胞菌的感染。制备锦鲤疱疹病毒 (KHV)的 2对引物KHV9/ 5F和KHV9/ 5R ,KHV1和KHV2 ,用嵌套式聚合酶链式反应 (nested -PCR)在脑和脾脏组织的抽提物中扩增出长度为 4 12bp的特异性的DNA片段。将该片段的nested -PCR扩增产物纯化后 ,克隆、测序。用NCBI -Blast软件将测序结果在Genebank中进行搜寻、比较 ,结果发现与注册号为AF4 1180 3的KHV基因序列的一部分片段有 99%的同源性。因此初步判断这次锦鲤暴发性疾病是由KHV引起的 。

Nested PCR检测微量隐孢子虫卵囊

根据隐孢子虫 18S r DNA序列 ,设计出隐孢子虫属和鼠隐孢子虫种的特异性引物 ,进行 PCR和 Nested PCR反应 ,先后分别扩增出 1条 5 4 0 bp和 1条 2 5 0 bp的条带。研究表明 ,Nested PCR具有高度的特异性和敏感性。应用Nested PCR可检测 1～ 10个鼠隐孢子虫卵囊 ,其敏感性是饱和蔗糖漂浮法的 10 5倍以上。

解脲脲原体套式（Nested）PCR检测研究

本文报告解脲脲原体（ＵＵ）的套式（ＮＥＳＴＥＤ）ＰＣＲ检测方法。经方法学考核表明，本法的特异性、灵敏度以及试剂的稳定性和对临检标本的顺应性均较好。９３份各种生殖道炎症患者之宫颈拭子标本套式ＰＣＲ检出２１份阳性（阳性率２２．６％），而市售ＰＣＲ试剂盒仅检出１份阳性（阳性率１．１％）。前者阳性检出率明显高于后者（Ｐ＜０．０１）。