RT-PCR技术诊断猪瘟的应用研究

应用反转录—聚合酶链反应(RT-PCR)对猪瘟进行诊断应用研究。应用RT-PCR况对来自广西不同地区的135份疑似猪瘟病料进行检测，以份诊断为阳性，阳性率62．2％。从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份，经RT-PCR检测，37份为阳性，阳性率为13．4％。其中健康猪扁桃体带毒较高，246份扁桃体中有35份阳性，占14．2％。采自柳州健康猪的26份淋巴结材料全为阴性，只有邕宁县的1份健猪淋巴结阳性。结果表明，RT-PCR技术可应用于猪瘟的临床诊断。     

Quantification of MDR-1 gene expression in canine tissues by real-time reverse transcription quantitative polymerase chain reaction

MDR-1 gene product mediated multidrug resistance is thought to play a major role in the outcome of chemotherapy in some canine tumors, especially malignant lymphoma. In the present study, MDR-1 RNA expression in normal lymph node and liver tissue as well as in tumor biopsies from 23 dogs with lymphomas and two dogs with liver tumors was measured by real-time RT-quantitative PCR. MDR-1 gene expression was detected in all samples analyzed. Comparably high MDR-1 RNA levels were measured in all normal liver tissues, one of the lymphomas and a cholangiocarcinoma. MDR-1 expression levels in canine lymphomas were found to vary over a wide range with most tumors expressing relative low levels. Interestingly, gastrointestinal lymphomas expressed higher MDR-1 RNA levels than multicentric lymphomas (p = 0.03). In conclusion, real-time RT-quantitative PCR appears to be a suitable method for sensitive and quantitative determination of MDR-1 gene expression in canine normal and neoplastic tissues.     

金黄地鼠Dpl基因克隆及其组织特异性表达的研究

本文成功克隆了金黄地鼠Dpl(PrP-like protein doppel,Doppel)基因,并采用实时荧光定量PCR法对其组织特异性表达进行了研究.根据已发表家鼠Dpl基因序列设计引物,采用PCR直接扩增金黄地鼠(Ham.ster)的Dpl基因CDs区,序列测定和分析表明金黄地鼠的Dpl基因CDs区全长537 bp,编码178个氨基酸的前体蛋白,不存在基因多态性.经基因序列对比分析,金黄地鼠与其他10种属哺乳动物Dpl基因有较高同源性,均为70.8%以上,且与鼠科Dpl序列的同源性最高(86%).实时定量PCR结果表明,在所检测组织中,Dpl m.RNA在睾丸表达量最高,其次为脾脏、心脏、骨髓和脑,其在肝脏、肺脏、肾脏和肌肉中的表达低于检测水平;成年动物与未成年动物相比,在睾丸组织的表达量,成年鼠显著高于未成年鼠;在成年鼠脑组织检测不到Dpl m.RNA 的表达.本研究对金黄地鼠Dpl基因序列进行分析测定,并对其在不同组织的生理表达进行了定量,以期为其功能的进一步研究提供基础数据.     

RT-PCR检测猪繁殖障碍与呼吸道综合征病毒方法的建立与应用

作者参考国内外已发表猪繁殖障碍与呼吸道综合征病毒(PRRSV)的基因序列和PRRSV相关的RT—PCR检测方法报道，根据PRRSV高度保守、高特异性的NP蛋白基因设计了一对引物，通过对疫苗毒株、细胞分离株的检测，建立了PRRSVRT—PCR检测方法。进一步对40份临床样品进行了检测应用，结果有17份阳性样品，与临床症状和血清学结果相符。     

RT-PCR技术检测猪瘟病毒的应用研究

本试验应用反转录-聚合酶链式反应(RT-PCR)对猪瘟进行诊断应用研究.应用RT-PCR对来自广西不同地区的135份疑似猪瘟病料进行检测,84份诊断为阳性,阳性率62.2%.从百色、柳州地区等采集的健康猪扁桃体和淋巴结共276份,经RT-PCR检测,37份为阳性,阳性率为13.4%.其中健康猪扁桃体带毒较高,246份扁桃体中有35份阳性,占14.2%.采自柳州健康猪的26份淋巴结材料全为阴性,只有邕宁县的1份健猪淋巴结阳性.结果表明,RT-PCR技术可应用于猪瘟的临床诊断.     

RT-PCR检测猪瘟病毒方法的建立与应用

建立RT PCR检测猪瘟病毒的方法。根据已发表的猪瘟病毒E2基因 (囊膜糖蛋白gP55基因 )序列 ,设计合成了一对特异性引物 ,扩增片段的大小为 50 7bp ,用RT PCR技术对石门系标准株和 1 0株分离株进行检测。结果这对引物对标准株和 1 0株分离株均能扩增出与预期大小相符 50 7bpRT PCR产物 ,而对其他 6种猪病病原核酸的扩增结果为阴性。该RT PCR可检出 1 0 0pg的猪瘟病毒RNA模板 ,对人工感染猪不同组织样品进行检测 ,结果对白细胞抽提的核酸样品检出率最高为 1 0 0 % (2 4 / 2 4 ) ,其次为扁桃体、脾、肾 ,检出率为 83 3 % (2 0 / 2 4 ) ,再者为淋巴结 ,检出率为66 7% (1 6/ 2 4 )。对送检的 1 9份疑似猪瘟的病死猪病料组织进行RT PCR检测 ,结果有 1 6份样品为猪瘟病毒阳性。兔体交叉反应试验结果RT PCR阳性的 1 6份病料中 ,有 1 4份样品被判为含有猪瘟病毒 ,其他病料兔体交叉反应试验结果全为阴性     

Relative quantification of canine CD56 mRNA expression by real-time polymerase chain reaction method in normal tissues and activated lymphocytes

Real-time PCR was optimized for the quantification of canine CD56 mRNA expression. This study was conducted to easily quantify canine CD56 expression and to identify its expression in normal tissues, peripheral blood mononuclear cells and activated lymphocytes in dogs. This assay revealed the highest level of CD56 mRNA expression in the normal canine brain, followed by the lung, kidney and liver. CD56 mRNA expression level in peripheral blood mononuclear cells was considerably lower; among activated lymphocytes in vitro, CD56 mRNA expression was increased.     

Quantitative genotyping by amplifying the polymorphic sequences of Pre-Melanosomal Protein (PMEL17) gene using real-time polymerase chain reaction in chickens

1. This study was conducted to develop a quantitative genotyping system of chimaeric chickens by real-time PCR. 2. The polymorphisms in exons 7 and 11 of PMEL17 gene, which is one of the major genes affecting plumage colour, were identified from White Leghorn, Barred Plymouth Rock and Rhode Island Red chickens. 3. Quantitative genotyping was successfully performed by real-time PCR using polymorphic sequence-specific TaqMan Probes. 4. This methodology can support future research of germline chimaeric chickens as well as the application of germ cell transfer technique.     

Reference gene selection for reverse transcription quantitative polymerase chain reaction in chicken hypothalamus under different feeding status

This study was designed to investigate the stability of 10 candidate reference genes, namely ACTB, B2M, GAPDH, HMBS, LBR, POLR2B, RN18S, RPS17, TBP, and YWHAZ for the normalization of gene expression data obtained by quantitative real‐time polymerase chain reaction (qPCR) in studies related to feed intake of chicken. Samples were isolated from hypothalamus under three different nutritional status (ad libitum, fasted for 24 hr, fasted for 24 hr then refed for 2 hr). Five different algorithms were applied for the analysis of reference gene stability: BestKeeper, geNorm, NormFinder, the comparative ΔCt method, and a novel approach using multivariate linear mixed‐effects modelling for stable reference gene selection. TBP and POLR2B were identified as the two most suitable and B2M and RN18S as the two least stable reference genes for normalization. Despite our review, the current literature showing that RN18S is one of the most commonly used reference gene in chicken gene expression studies, its applicability for normalization should be evaluated before each qPCR experiment.