定量RT-PCR法对"苦参"在心肌炎模型中药效学研究

目的用定量RT-PCR法检测“苦参”中抗柯萨奇B病毒有效成份“抗柯注射液”,在Balb/c小鼠病毒性心肌炎模型病毒血症时,对抗“柯萨奇B病毒”的药效。 方法取48只雄性18-22g 8周龄的Balb/c小鼠,随机分成A-H共8组,每组6只。1.各治疗组(A-F组),每只小鼠从腹腔接种0.1ml l04TCID50 CVB3m病毒2h后,每天从尾静脉分别注射抗柯注射液,5、10、15、20、25、30mg/kg,2次/d,连续用药3d。2.病毒组(G组),注射病毒同上法,2h后从尾静脉注入0.3ml无菌生理盐水,2次/d,连续3d。3.空白对照组(H组),腹腔内注射无小牛血清的RPMI-1640培养液,2h后从尾静脉注入0.3ml无菌生理盐水,2次/d,连续3d。 结果 空白组CVB-RNA(-)对接种CVB3m感染后用不同剂量“抗柯注射液”治疗的各小组血液中CVB3m-RNA含量与病毒组相比明显下降,其抑制率(％)与所用抗柯注射液的剂量呈正比,即“抗柯”剂量增加,CVB3m-RNA含量下降的幅度亦增加,每天5-30mg,/kg中的各剂量都有明显抑制病毒增殖的作用。 结论用定量RT-PCR法检测到“抗柯注射液”对Balb/c小鼠病毒性心肌炎病毒血症时效果明显,最小有效剂量为5mg·kg-1·d-1。     

采用通用引物PCR与机器码识别技术快速鉴定血液感染病原菌

目的 建立一种快速检测血液感染病原菌的方法.方法 选取7种常见血液感染病原菌,在其16S rRNA末端和23S rRNA始端的保守区设计通用引物,对16S-23S rRNA基因间区进行扩增,然后将PCR产物的电泳图谱进行机器码识别,以鉴定病原菌.结果 通用引物能扩增出7种病原菌,且每种菌具有不同片段长度(bp):大肠埃希菌(716、808),金黄色葡萄球菌(732、827),铜绿假单胞菌(833),表皮葡萄球菌(630、722、817),肺炎链球菌(606),肺炎克雷伯菌(550、705、797),粪肠球菌(591、693);此图谱可经机器码识别,对各种病原菌进行快速鉴定.结论 采用通用引物PCR和机器码识别技术可简便、快速、特异地鉴定常见血液感染病原菌.     

蛋白激酶C亚型在血管外膜成纤维细胞中的表达

目的了解蛋白激酶C（PKC）亚型在血管外膜成纤维细胞中的表达情况。方法用β1-转化生长因子（TGF-1β）可将AF诱导为MF,用免疫组化法可以鉴定转化是否成功。根据文献报道,蛋白激酶C的许多亚型在AF与MF中的表达量往往差异有统计学意义。实验选取PKC-ε、PKC-δ、PKC-ζ作为研究对象用定量PCR的方法,比较它们在AF和MF中表达的差异。结果 PKC-ε、PKC-ζ的表达在MF中明显高于AF,而PKC-δ的表达差异无统计学意义。结论本实验为进一步研究PKC家族在血管外膜成纤维细胞表型转化中的作用打下基础。     

National Scale Real-Time Surveillance of SARS-CoV-2 Variants Dynamics by Wastewater Monitoring in Israel

In this report, we describe a national-scale monitoring of the SARS-CoV-2 (SC-2) variant dynamics in Israel, using multiple-time sampling of 13 wastewater treatment plants. We used a combination of inclusive and selective quantitative PCR assays that specifically identify variants A19/A20 or B.1.1.7 and tested each sample for the presence and relative viral RNA load of each variant. We show that between December 2020 and March 2021, a complete shift in the SC-2 variant circulation was observed, where the B.1.1.7 replaced the A19 in all examined test points. We further show that the normalized viral load (NVL) values and the average new cases per week reached a peak in January 2021 and then decreased gradually in almost all test points, in parallel with the progression of the national vaccination campaign, during February–March 2021. This study demonstrates the importance of monitoring SC-2 variant by using a combination of inclusive and selective PCR tests on a national scale through wastewater sampling, which is far more amendable for high-throughput monitoring compared with sequencing. This approach may be useful for real-time dynamics surveillance of current and future variants, such as the Omicron (BA.1, BA.2) and other variants.     

巢氏PCR扩增强直性肌营养不良基因在植入前诊断中的局限性

目的I型强直性肌营养不良(myotonic dystrophy 1,DM1)是由于肌强直蛋白激酶基因(DMPK)3`端非翻译区的(CTG)n异常扩增导致。探讨用巢氏PCR法直接扩增该基因在植入前诊断的可行性。方法0.5%低熔点琼脂糖分离来自DMPK(CTG)100的DM1患者的单个精子,巢氏PCR法扩增DMPK基因。结果106例精子标本中53例扩增产物电泳为单条带,但48例产物显示为多条带,扩增产物长度介于(CTG)10至(CTG)30之间。结论利用低熔点琼脂糖可有效分离单个精子进行检查,而巢氏PCR在单细胞水平上直接扩增较多(CTG)n的DMPK基因时存在困难,应用于植入前诊断时容易造成误诊。     

Detection of Gyrovirus galga 1 in Cryopreserved Organs from Two Commercial Broiler Flocks in Japan

Gyrovirus galga 1 (GyVg1, previously recognized as avian gyrovirus 2), which was first reported in chicken in 2011, is a new member of the genus Gyrovirus. The presence of GyVg1 has also been confirmed in different regions of Europe, South America, Africa, and Asia, indicating its global distribution. However, because there are no reports of examining the distribution of GyVg1 in animals in Japan, the epidemiology of this virus is unknown. In this study, we attempted to retrospectively detect GyVg1 in cryopreserved chicken materials derived from different two commercial broiler flocks in 1997. The GyVg1 genome was detected in organ materials derived from both flocks by PCR. GyVg1 detected in both flocks was classified into four genetic groups by analyzing the nucleotide sequences of the detected PCR products. These results suggest that diverse GyVg1 strains were present in commercial chicken flocks as early as 1997 in Japan.     

Detection of immunoglobulin gene rearrangement in leukemia by seminested PCR amplification and Southern blot hybridization

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C6/36细胞系分离登革1型病毒的方法学探讨

目的应用C6/36细胞系3种细胞分离方法分离血清中登革1型病毒并探讨其分离效率。方法采用血清预稀释法,将已鉴定为登革I型病毒的阳性血清作10^-1-10^-10等10个10倍梯度稀释并接种于长满单层C6/36细胞的培养管;采用共培养法,将血清作50至6400等8个梯度倍比稀释,再加等体积C6/36细胞悬液作共培养;采用直接接种法,将25μl、50μl、100μl、150μl、200μl血清接种于长满单层C6/36细胞的培养管。连续7d,每天观察细胞生长情况,收获细胞分离液并提取核酸,然后采用Real-time PCR检测其CT值。结果血清标本呈登革I型病毒阳性,CT值为26.3;血清预稀释法与共培养法均未发现由血清毒性引发的非特异性细胞病变,除了10^-10稀释度呈弱阳性外,其余稀释度的CT值均在11-15之间。直接接种法中25μl、50μl接种量培养管未发现由血清毒性引起明显的非特异性细胞病变,检测其CT值约为19,100μl、150μl、200μl接种量的细胞第2d就因血清毒性完全被破坏。结论3种方法都能成功分离登革1型病毒,降低血清对细胞的干扰作用能提高分离效率。