SYBR Green Ⅰ实时荧光定量RT-PCR测定猴免疫缺陷病毒RNA拷贝数方法的建立

.学者论坛·在动物实验中解决临床难题···························，·······································································……顾玉东(3)基因〕:程     

Real-time nested multiplex PCR for the detection of herpes simplex virus types 1 and 2 and varicella zoster virus

One hundred forty-nine specimens were tested in a LightCycler nested multiplex polymerase chain reaction (LCnmPCR) for Herpes simplex virus (HSV)1, HSV2, and VZV. Eighty-one were from genitourinary medicine (GUM) patients and the other 68 specimens were from other patients with skin lesions. The results were compared to a conventional multiplex nested PCR (nmPCR) using agarose gel electrophoresis. Twenty-five specimens were positive in both assays for HSV1 and 29 were positive for VZV. For HSV2 there were 27 positive in the LCnmPCR and 26 positive in the nmPCR assay. The melting temperatures (Tms) of each target were different with a mean of 84.75 degrees C for HSV1, 88.57 degrees C for HSV2, and 83.62 degrees C for VZV. The melting curves of positive specimens directly overlaid the melting curves of the positive controls in the assay. The LCnmPCR assay is a convenient alternative to conventional PCR using agarose gel electrophoresis. It improves specimen turnaround time by eliminating the need for gel electrophoresis, transillumination, and gel photography. It also shows increased sensitivity for HSV2 over our standard assay. This LCnmPCR reduces further the possibility of amplicon contamination with nested PCR protocols.     

Rapid identification of female carriers of DMD/BMD by quantitative real-time PCR

Recently developed PCR systems offer online-monitoring of amplification and allow simple and reliable DNA quantification. We have used the LightCycler system to develop a simple and rapid method for direct identification of female carriers of deletions and duplications in the dystrophin gene. The challenge resides in the ability to identify the presence of a deleted or duplicated allele over the background contributed by the normal allele. Quantification is based on the determination of the ratio between potentially deleted/duplicated dystrophin exons and non-deleted/-duplicated reference exons using the unspecific dsDNA-dye SYBRgreen I. In a retrospective study, we evaluated our method in female relatives of DMD/BMD patients with known carrier status by comparative analysis of deleted or duplicated versus non-deleted/-duplicated exons. Carrier status was accurately attributed in 100% of cases, the mean ratios being 0.52+/-0.12 for deletion carriers (expected value: 0.5) and 1.56+/-0.18 for duplication carriers (expected value: 1.5) vs. 1.022+/-0.17 for non-carriers (expected value: 1.0). The method proved to be simple, rapid, reliable, and cost-effective. It may be used for direct determination of deletions/duplications in potential DMD/BMD carriers and may easily be adapted for other genetic conditions involving deletions and duplications.     

Regulation of cell growth and expression of 7B2, PC2, and PC1/3 by TGFbeta 1 and sodium butyrate in a human pituitary cell line (HP75)

Recent studies have shown that 7B2 and the neuroendocrine-specific proconvertase PC2 have important roles in pituitary cell proliferation and hormone secretion. Studies from our laboratory have also shown that TGFb1 regulates anterior pituitary cell proliferation and hormone secretion. To study the regulation of 7B2 in human pituitary tumors, we used a cell line derived from a human pituitary adenoma (HP75) that has been shown to express 7B2, PC1, PC2, and TGFβ receptors to analyze the effects of TGFβ1 and the histone deacetylase inhibitor (HDACI) sodium butyrate (NaB) treatment on 7B2 mRNA expression along with the neuroendocrine-specific proconvertases 1/3 (PC1) and PC2 mRNA and protein expression. RNA was quantified by real-time PCR and proteins were detected by immunohistochemistry and Western blotting. Treatment of cells with 1 mM NaB or 1 nM TGFβ1 for 4 d decreased cell proliferation with a concomitant increase in the cell cycle protein p21. Real-time PCR analysis showed a significant increase in 7B2 mRNA after NaB and TGFβ1 treatment. PC2 mRNA was down regulated by NaB while PC1 mRNA was unchanged. TGFβ1 stimulated PC1, but not PC2 mRNA levels. Changes in PC1 and PC2 protein were similar to changes in the mRNAs, but the differences were not significant. These results indicated that NaB and TGFβ1 inhibit pituitary cell proliferation and regulate the expression of 7B2, PC1, and PC2 in a cell culture model of pituitary tumors. Our results also indicate that inhibition of pituitary cell proliferation is associated with increased expression of 7B2 mRNA.     

The Potential of β Carbolin Alkaloids to Hinder Growth and Reverse Chloroquine Resistance in Plasmodium falciparum

Background:

Nowadays, scourge of malaria as a fatalistic disease has increased due to emergence of drug resistance and tolerance among different strains of Plasmodium falciparum. Emergence of chloroquine (CQ) resistance has worsened the calamity as CQ is still considered the most efficient, safe and cost effective drug among other antimalarials. This urged the scientists to search for other alternatives or sensitizers that may be able to augment CQ action and reverse its resistance.

Method:

Three β-carbolin derivatives, namely, harmalin, harmol and harmalol were tested for their anti-plasmodial and CQ resistance reversal effects against P. falciparum 3D7 and K1. SYBRE Green-1 based drug sensitivity assay and isobologram analysis were used to screen the mentioned effects respectively.

Results:

All of them showed moderate anti-plasmodium effect and harmalin was the most effective as compared to the others in reversing CQ resistance and tolerance.

Conclusion:

The mentioned phytochemicals are not ideal to be used as conventional antimalarials and only harmalin can be suggested to reverse CQ resistance in P. falciparum K1.     

应用SYBR Green I荧光定量RT-PCR测定糖尿病大鼠肾皮质TGF-β1表达水平

SYBR GreenI荧光定量RTPCR法检测正常、糖尿病和牛磺酸治疗组大鼠TGFβ1mRNA/βactinmRNA比值为(7.0±0.8)×10-3,(64.4±8.0)×10-3和(16.7±2.0)×10-3;终点法RTPCR检测比值分别为0.28±0.12,0.58±0.16和0.43±0.10。与终点法RTPCR相比,TGFβ1mRNASYBRGreenI荧光定量RTPCR实时检测法,方便快捷,敏感特异。     

一种基于ELISA检测氮芥诱导的MGMT-DNA交联体的方法

目的 建立一种基于酶联免疫吸附法检测O6-烷基转移酶(O6-Methylguanine-DNA methyltransferase,MGMT)与DNA的交联体(MGMT-DNA crosslink,M-DPC)含量的简便方法.方法 整合当前技术提取及纯化M-DPC、定量M-DPC样品中dsDNA含量、优化DPC中抗原在ELISA板的包被能力等关键环节.以不同浓度氮芥处理人支气管上皮细胞HBE,在指定的时间收集细胞,使用细胞核抽提/裂解/Tris饱和酚回收法制备M-DPC样品.采用SYBR Green Ⅰ染色方法对M-DPC样品中dsDNA进行定量.采用全能核酸酶对M-DPC样品中脱氧核糖核酸降解,以增强M-DPC样品中抗原在ELISA板的包被能力.最终,包被后的多孔板检测采用常规的ELISA.结果 无需高速离心机和放射性同位素等,使用常规方法提取了细胞M-DPC.SYBR Green Ⅰ检测基因组dsDNA的线性范围为2 ng至1.5 μg,可以用于提取的M-DPC所含dsDNA的定量,据此统一样本用量.M-DPC可以通过ELISA检出,且在一定范围内有线性关系.氮芥染毒3h后M-DPC含量有显著增加,且对氮芥剂量有依赖.至24 h M-DPC含量无显著降低.结论 所建立的ELISA检测M-DPC的方法快速、经济、灵敏、高通量,也可应用于部分其他交联蛋白的检测.氮芥染毒所致M-DPC形成有时间和剂量效应.     

检测蚊虫感染黄病毒属病毒Real-time PCR方法的建立

目的 建立SYBR Green Real-time PCR检测和筛选黄病毒属病毒方法. 方法 参照文献报道的通用引物,以JEV cDNA 和DEN cDNA为模板,建立RT-PCR和SYBR Green Real-time PCR,检测和筛选黄病毒属病毒,并比较两者的敏感性. 结果 此黄病毒属引物适合两种反应体系,SYBR Green Real-time PCR方法的敏感性是RT-PCR方法的100倍,最低检出病毒浓度为0.5×10-2 PFU/ml. 结论 建立的两种方法均可用于黄病毒属病毒检测,以SYBR Green Real-time PCR方法具有更高的敏感性,对于黄病毒属病毒初筛检测具有应用价值.     

建立染料法荧光定量PCR检测恙虫病东方体

目的建立染料法荧光定量PCR检测恙虫病东方体。方法根据恙虫病东方体56kD外膜蛋白基因序列设计特异性引物,建立染料法(SYBR green I)实时荧光定量PCR,评估其灵敏性及特异性;并对恙虫病东方体鸡胚培养物、东方体感染小白鼠脏器标本以及恙虫病病人血样等多种样本进行检测。结果建立的荧光定量PCR标准曲线的循环阈值(Ct)与模板拷贝数呈良好的线性关系(r2=0.99),灵敏性评估显示20μl体系单PCR反应管靶基因大于28个拷贝即可被有效检测,最低检出浓度为2拷贝/μl,比普通PCR检测方法提高1~2个数量级,并且具有较好的重复性;建立的SYBR I染料法具有良好的特异性,与立氏立克次体R株等其他5种立克次体,以及被检测的其他几种病原菌DNA模板均不发生特异性扩增;用建立的染料法对东方体鸡胚培养物、东方体感染动物脏器,以及采集的现场恙虫病病人血样共59份标本进行检测,其中57份检出阳性。用该定量PCR检测恙虫病东方体实验感染小鼠的血、脾脏、肾脏、肝脏标本,结果脾脏中东方体检出量最多,肝脏和肾脏次之,血中的恙虫病东方体量较低。结论本研究建立荧光定量PCR方法均具有很高的特异性和敏感性,适合各种样本中东方体的快速检测,可用于实验室的快速诊断。