凝胶成像系统对核酸扩增产物的定量检测

目的 建立一种PCR-凝胶成像系统定量分析核酸扩增产物的方法．方法：1．用PCR荧光定量分析仪实时监测PCR扩增曲线，并选择合适的扩增循环次数，2．将梯度标准血清(10^1～10^6拷贝／u1)分别进行40，35及32次循环次数的扩增，在琼脂糖电泳，EB染色后，进行凝胶分析；3．选择凝胶分析软件中能客观反映核酸实际含量的最适参数，制定标准曲线。结果:40及35次循环时，在10^1～10^4拷贝／ul，线性良好，而在10^4，10^5及10^6拷贝／ul三个梯度时直线上升缓慢，趋于平坦；32次循环时，10拷贝／ul未能检出，10^2～10^6拷贝／ul的5个梯度的模板(对数值)与产物(体积)有良好的线性关系(r=0．97)。结论：32次循环线性范围较广，为最适循环次数；在UVIband的光密度定量分析指标中，以体积或体积百分比作为定量指标较好；本法除可用于常规PCR产物定量(HBV-DNA，TB—DNA)，也适用于RT-PCR(HCV-RNA)。

凝胶图像处理系统定量检测乙型肝炎病毒DNA

目的建立一种聚合酶链反应 (PCR) -凝胶图像处理系统定量分析核酸扩增产物的方法。方法 1.用PCR荧光定量分析仪实时监测 PCR扩增曲线 ,并选择合适的扩增循环次数 ;2 .将梯度标准乙型肝炎病毒 (HBV )阳性血清 (10 1 ～ 10 6 拷贝 /μl)分别进行 40 ,35及 32次循环次数的扩增 ,经琼脂糖电泳进行凝胶分析 ;3.选择凝胶分析软件中能客观反映核酸实际含量的最适参数 ,制定标准曲线。结果 40及 35次循环时 ,在 10 1 ～ 10 4拷贝 /μl线性良好 ,而在 10 4,10 5 及 10 6 拷贝 /μl 3个梯度时直线上升缓慢 ,趋于平坦。 32次循环时 ,10拷贝 /μl未能检出 ,10 2～ 10 6拷贝 / μl的 5个梯度的模板 (对数值 )与产物 (体积 )有良好的线性关系 (r=0 .97)。结论 32次循环线性范围较广 ,为最适循环次数 ;在 U VI band的吸光度定量分析指标中 。

Novel biosensor-based microarray assay for detecting rs8099917 and rs12979860 genotypes

AIM:To evaluate a novel biosensor-based microarray(BBM) assay for detecting rs12979860 and rs8099917 genotypes.METHODS:Four probes specific for rs8099917C/T or rs12979860G/T detection and three sets of quality control probes were designed,constructed and arrayed on an optical biosensor to develop a microarray assay.Two sets of primers were used in a one tube polymerase chain reaction(PCR) system to amplify two target fragments simultaneously.The biosensor microarray contained probes that had been sequenced to confirm that they included the rs8099917C/T or rs12979860G/T alleles of interest and could serve as the specific assay standards.In addition to rehybridization of four probes of known sequence,a total of 40 clinical samples collected from hepatitis C seropositive patients were also tested.The target fragments of all 40 samples were amplified in a 50 μL PCR system.Ten μL of each amplicon was tested by BBM assay,and another 40 μL was used for sequencing.The agreement of the results obtained by the two methods was tested statistically using the kappa coefficient.The sensitivity of the BBM assay was evaluated using serial dilutions of ten clinical blood samples containing 10 3-10 4 white cells/μL.RESULTS:As shown by polyacrylamide gel electrophoresis,two target segments of the interleukin 28Bassociated polymorphisms(SNPs) were successfully amplified in the one-tube PCR system.The lengths of the two amplified fragments were consistent with the known length of the target sequences,137 and 159 bps.After hybridization of the PCR amplicons with the probes located on the BBM array,the signals of each allele of both the rs8099917 SNPs and rs12979860 SNPs were observed simultaneously and were clearly visible by the unaided eye.The signals were distinct from each other,could be interpreted visually,and accurately recorded using an ordinary digital camera.To evaluate the specificity of the assay,both the plasmids and clinical samples were applied to the microarray.First,30 PCR amplicons of the various SNP alleles were hybridized on the BBM microarray.Full agreement between plasmids and the BBM assay was observed,with 30/30 correct matches(100%).The kappa value for the BBM assay with plasmids was 1.00(P < 0.05).For the 40 clinical blood samples,the BBM assay hybridization and direct sequencing results were compared for each amplicon.For patient blood samples,agreement was 28/28 for rs8099917T/T,9/11 for rs8099917T/G,1/1 for rs8099917G/G,24/24 for rs12979860C/C,11/14 for rs12979860C/T,and 2/2 for rs12979860T/T.Only five clinical samples of amplicon assay and direct sequencing results were discordant and heterozygotes:2/11 rs8099917T/G and 3/14 rs12979860C/T.The agreement of outcomes between BBM assay and direct sequencing for the detection of rs8099917 and rs12979860 was 95% and 92.5%,respectively;and the corresponding kappa values were 0.88 and 0.85(A kappa value > 0.75 was defined as substantial agreement).The BBM assay and sequencing had similar specificities for detection and identification of the two SNPs and their alleles.The sensitivity evaluation showed that the BBM assay could detect and identify SNP sequences present in blood samples containing as few as 10 2 white blood cells/μL.CONCLUSION:This biosensor microarray assay was highly specific,sensitive,rapid and easy to perform.It is compatible with clinical practice for detection of rs8099917 and rs12979860.

RT—PCR—ELISA方法检测甲肝减毒活疫苗病毒滴度的初步研究

目的应用核酸扩增产物测定的固相杂交酶联显色法(RT-PCR-ELISA)检测甲肝减毒活疫苗病毒滴度。方法应用RT-PCR-ELISA，将标记有生物素的寡核苷酸引物所扩增的疫苗病毒基因产物，与微孔反应板上的特异性探针进行快速杂交，通过辣根过氧化物酶标记的链亲和素进行酶联显色，读取吸光度(A值)，判断结果。应用此法检测了11批甲肝活疫苗滴度，并与常规细胞培养法(CCID50）比较。结果本方法与细胞培养法的敏感性相仿，具有简便、快速、特异的优点。结论RTPCR—EUSA法有望代替常规细胞培养法应用于甲肝减毒活疫苗病毒滴度的检测。

基于PCR技术可视化鉴别金钱白花蛇方法的建立

目的 基于聚合酶链式反应(PCR)技术,利用核酸扩增产物,建立一种可以快速鉴定金钱白花蛇中药材真伪的可视化试纸条检测方法。方法 提取金钱白花蛇、赤链蛇及市售样本的基因;根据mtDNA Cytb序列进行对比分析,设计1对特异性鉴别引物,进行修饰物标记;建立鉴别PCR反应体系,使用核酸扩增产物,利用可视化试纸条对市售样本进行真伪鉴定。结果 鉴别引物可将正品金钱白花蛇中药材扩增出500~600 bp之间的特异性条带,而伪混品则不出现相应的特异性条带;可视化试纸条对市售样本进行检测,鉴别效果良好,灵敏度高,可准确鉴定中药材真伪。结论 可视化试纸条可以快速鉴别金钱白花蛇中药材真伪,稳定性高、特异性强,可应用于市场出售金钱白花蛇中药材的鉴定。