复合探针实时荧光RT-PCR法检测小儿上呼吸道感染甲型流感病毒的价值

目的 分析小儿上呼吸道感染甲型流感病毒应用复合探针实时荧光反转录聚合酶链反应(RT-PCR)法检测的临床价值。方法选择2023年1月至2023年12月流感监测信息系统两家监测点上呼吸道感染甲型流感病毒感染的患儿80例监测标本进行回顾性分析,均开展复合探针实时荧光RT-PCR法检测,分析其诊断价值。结果 根据监测标本最终诊断结果显示,阳性标本68例、阴性标本12例。经复合探针实时荧光RT-PCR法检出67例,检出率为83.75%,敏感度为95.59%、特异度为83.33%、准确度为93.75%、阳性结果预测值为97.01%、阴性结果预测值为76.92%;批间批内变异系数均小于5%。结论 小儿上呼吸道感染甲型流感病毒应用复合探针实时荧光RT-PCR技术具有较高的敏感度、特异度及准确度,且检查结果快速,可为小儿上呼吸道感染甲型流感病变提供可靠的诊断,有利于制订合理的治疗方案。

Detection of circulating hepatocellular carcinoma cells in peripheral venous blood by reverse transcription-polymerase chain reaction

Objective: To detect circulating hepatocellular carcino-ma by demonstrating hepatocellular carcinoma cells orhepatocyte-associated mRNA in the nuclear cell com-ponent of peripheral blood (PBL).Methods: Peripheral blood (5 ml) samples were ob-tained from 93 patients with hepatocellular carcinoma(HCC) and from 33 control subjects (9 with liver cir-rhosis after hepatitis B,14 with chronic hepatitis B,10with normal liver function). To identify HCC cells inperipheral blood, liver-specific human alpha-fetopro-tein (AFP) mRNA was amplified from total RNA ex-tracted from whole blood by reverse transcription-polymerase chain reaction.Results: AFPmRNA was detected in 50 blood samplesfrom the HCC patients (50/93, 53.8%). In contrast,there were no clinical control patients whose samplesshowed detectable AFPmRNA in PBL. The presence ofAFPmRNA in blood seemed to be correlated with thestage (by TNM classification) of HCC, the serum AFPvalue, and the presence of intrahepatic metastasis,portal vein thrombosis, tumor diameter and/or distantmetastasis. In addition, AFPmRNA was detected in theblood of 21 patients with metastasis at extrahepaticorgans (100%) in contrast to 29 (40.3%)of 72 pa-tients without metastasis.Conclusion: The presence of AFPmRNA in peripheralblood may be an indicator of malignant hepatocytes,which might predict hematogenous spreading metasta-sis of tumor cells in patients with HCC.

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.

帕利亚姆病毒实时荧光定量RT-PCR检测方法的建立与应用

本研究拟建立帕利亚姆病毒(Palyam virus,PALV)血清型特异性实时荧光定量RT-PCR(qRT-PCR)方法用于临床样本或媒介中PALV血清型鉴定。根据我国流行PALV毒株的基因节段2序列,设计扩增引物和TaqMan探针,建立PALV血清型特异型qRT-PCR方法,对方法的特异性、灵敏性与重复性进行评估;以我国分离的28株PALV和90份核酸阳性血液样本评估检测方法的可靠性;利用建立的方法对采集库蠓样本中携带的PALV进行血清型鉴定。结果显示,建立的PALV血清型qRT-PCR检测方法具有良好的特异性与灵敏性,可检出核酸拷贝数下限在22至28 copies·μL^(-1)。对28株PALV的qRT-PCR检测结果与病毒测序鉴定结果一致;对PALV不同感染阶段哨兵动物血液(90份)中的qRT-PCR鉴定结果与分离病毒的血清型鉴定结果一致;建立的方法可准确鉴定库蠓中携带PALV的血清型。本研究建立的PALV血清型qRT-PCR定型方法具有良好的特异强、敏感性与重复性,可用于PALV感染动物与媒介中PALV血清型的鉴定,具有良好的应用价值。

Quantification of Porcine Follicle-stimulating Hormone Receptor Messenger Ribonucleic Acid by Reverse Transcription competitive Polymerase Chain Reaction

An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.

Exogenous reference gene normalization for real-time reverse transcription-polymerase chain reaction analysis under dynamic endogenous transcription

Quantitative real-time reverse transcription-polymerase chain reaction （qPCR） is widely used to investigate transcriptional changes following experimental manipulations to the nervous system. Despite the widespread utilization of qPCR, the interpretation of results is marred by the lack of a suitable reference gene due to the dynamic nature of endogenous transcription. To address this inherent deficiency, we investigated the use of an exogenous spike-in mRNA, luciferase, as an internal reference gene for the 2ct normalization method. To induce dynamic transcription, we systemically administered capsaicin, a neurotoxJn selective for C-type sensory neurons expressing the TRPV-1 receptor, to adult male Sprague-Dawley rats. We later isolated nodose ganglia for qPCR analysis with the reference being either exogenous luciferase mRNA or the commonly used endogenous reference 13-111 tubulin. The exogenous luciferase mRNA reference clearly demonstrated the dynamic expression of the endogenous reference. Furthermore, variability of the endogenous reference would lead to misinterpretation of other genes of interest. In conclusion, traditional reference genes are often unstable under physiologically normal situations, and certainly unstable following the damage to the nervous system. The use of exogenous spike-in reference provides a consistent and easily implemented alternative for the analysis of qPCR data.

基于直扩RT-PCR技术的寨卡病毒快速检测方法的建立

目的建立基于直扩实时荧光定量逆转录聚合酶链反应(RT-PCR)技术的寨卡病毒快速检测方法。方法采用特殊功能的DNA聚合酶,以及优选PCR增强剂,以此建立直扩RT-PCR技术的寨卡病毒快速检测5种样本(全血、血清、唾液、咽拭子和尿)的方法。结果5种样本检测限分别为血清103 PFU/mL,尿、咽拭子和唾液102 PFU/mL,全血104 PFU/mL,标准曲线的拟合优度的可决系数均在0.98以上,扩增效率均在90%~110%;寨卡病毒核酸成功扩增,非寨卡病毒核酸均未能扩增;尿、全血和唾液样本的重复性实验中106 PFU/mL和102 PFU/mL两个浓度的6个重复Ct值的变异系数均<5%。该研究建立的直扩RT-PCR技术的寨卡病毒检测方法与常规RT-PCR技术的检测结果一致,8个寨卡病毒样本,均只检测出2个血清样本,其余62个非寨卡病毒样本及12个阴性样本均未得到扩增。结论成功建立基于直扩RT-PCR技术的寨卡病毒快速检测方法,该方法简便快捷且灵敏度高、特异度强。

RT-PCR法在沙门菌检测及血清分型鉴定中的应用价值研究

目的:探讨反转录-聚合酶链反应(RT-PCR)法在沙门菌检测及血清分型鉴定中的应用价值。方法:选择2022年8月至2024年1月本疾控中心收治的456例腹泻患者的粪便标本,均行细菌培养和RT-PCR检测,记录RT-PCR鉴定沙门菌血清分型结果,以细菌培养结果作为金标准,分析RT-PCR鉴定沙门菌的价值及其与细菌培养的一致性,对比细菌培养、RT-PCR鉴定沙门菌的费用及操作时间。结果:456例腹泻中经细菌培养共检出沙门菌78株,RT-PCR检出85株,RT-PCR检测敏感度为97.44%(76/78),特异度为96.83%(366/378),准确度为96.93%(442/456),阳性预测值为89.41%(76/85),阴性预测值为99.46%(366/368);RT-PCR鉴定沙门菌结果与细菌培养一致性较高(kappa值=0.897,P<0.001);RT-PCR鉴定结果与细菌培养完全一致有73株,符合率为93.59%,其中里森沙门菌、鼠伤寒沙门菌、德尔卑沙门菌、姆班达卡沙门菌4种优势血清型结果完全一致,而与细菌培养鉴定不一致菌株5株;RT-PCR鉴定沙门菌操作时间为(0.30±0.07)天,费用(100.22±6.52)元,细菌培养鉴定沙门菌的操作时间为(2.82±0.25)天,费用为(300.52±12.16)元,RT-PCR鉴定沙门菌的费用及操作时间均低于细菌培养,差异有统计学意义(P<0.001)。结论:RT-PCR法可准确检出沙门菌,判断沙门菌血清分型,与细菌培养结果具有较高的一致性,且操作用时短、所需费用低,可应用于沙门菌检测。

Detection of hepatocellular carcinoma cells in the peripheral blood with reverse--transcription polymerase chain reaction

In order to detect circulating cells of hepatocellular carcinoma(HCC) in the peripheral blood with reverse transcripition polymerase chain reaction (RT-PCR ), alpha-fetoprotein (AFP ) mRNA was tested in the blood samples of 113 cases of HCC and 69 controls (including 30 cases of liver cirrhosis, 9 cases of metastatic liver cancer and 30 normal subjects). 20/43 (46. 5% ) cases of HCC and 2/30 (6. 7% ) cases of liver cirrhosis are positive and the cases of nletastatic liver cancer and normal controls were negative for human AFP(hAFP) rnRNA. The presence of hAFP mRNA in the peripheral blood seems to be correlated with intrahepatic and distant nletastasls of HCC and portal vein thrombosis. It is concluded that the presence of hAFP mRNA in the peripheral hloocl is an indicator of circulating HCC cells and can be used to diagnose the rnetastasisof HCC through henlatogenous route and RT-PCR amplification of hAFP mRNA is a sensitive and specificprocedure for detecting circulating cells of HCC.

猪德尔塔冠状病毒RT-nPCR检测方法的建立与N基因变异分析

建立一种基于猪德尔塔冠状病毒(PDCo V)N基因的RT-n PCR检测方法,对陕西省猪群PDCo V感染情况进行流行病学调查,分析PDCo V流行毒株N基因变异情况。通过病毒分离、检测引物设计、反应体系与条件优化、灵敏性试验和特异性试验,建立了PDCo V RT-nPCR检测方法,并对N基因片段进行序列分析。结果表明:建立的RT-n PCR方法能够扩增出303 bp的N基因目的片段,检测单链c DNA的极限为2.0194×10^(-3)ng·μL^(-1),该方法检测PEDV、TGEV、PRo V、CSFV与PRRSV均无交叉反应,特异性良好;陕西省猪场184份样品检测结果显示,PDCo V阳性率为6.52%(12/184);N基因序列分析发现,获得的HY2019、QLS2020的N基因片段同源性为99.0%,提示可能为同一来源;进化树分析发现,HY2019、QLS2020与我国四川株SCNC201705、Sichuan2017、Sichuan2019同处于一个小的进化分支上,且均存在168~179位“TGTCTGTCTCTA”共12个核苷酸缺失,推测陕西株与四川株高度同源,可能起源于早期四川毒株。

荧光定量RT-PCR法在病原微生物检验中的应用价值

目的:分析荧光定量逆转录聚合酶链式反应(RT-PCR)法在病原微生物检验中的应用价值。方法:选取2019年4月-2021年1月在长沙市中心医院进行病原微生物检测的46例疑似微生物感染患者作为研究对象,均实施常规RT-PCR法、荧光定量RT-PCR法检测,统计两种方法的检验结果;以特殊染色镜检结果作为“金标准”,比较两种检验方法检测微生物感染的准确度、灵敏度、特异度以及对各类病原微生物的检出率,统计受试者对两种检验方法的满意度。结果:特殊染色镜检结果显示,46例受试者病原微生物感染阳性43例,阴性3例。两种检验方法检测微生物感染的准确度、特异度比较,差异无统计学意义(P>0.05);荧光定量RT-PCR法检测灵敏度高于RT-PCR法,差异有统计学意义(P<0.05)。特殊染色镜检结果显示,43例病原微生物感染患者中,衣原体感染7例,支原体感染8例,寄生虫感染9例,细菌感染7例,真菌感染5例,病毒感染7例,两种检验方法对各类病原微生物的检出率及总检出率比较,差异无统计学意义(P>0.05)。受试者对荧光定量RT-PCR法检验总满意度高于RT-PCR法,差异有统计学意义(P=0.026)。结论:荧光定量RT-PCR法在病原微生物检验中的应用价值较好,能够提高检测灵敏度,受试者满意度高。

柯萨奇病毒A组5型实时荧光RT-PCR检测方法的建立与评价

目的建立柯萨奇病毒A组5型(CVA5)特异的一步法实时荧光反转录聚合酶链反应(RT-PCR)核酸检测方法。方法以流行于遵义地区的CVA5病毒株和NCBI基因库中随机下载的CVA5 VP1基因序列作为参考序列,分析这些序列的保守区域,在其保守区设计特异性的引物与Taqman探针,建立检测CVA5的一步法实时荧光RT-PCR。通过构建含CVA5 VP1保守区域的重组质粒,绘制标准曲线,并对检测方法的灵敏度、特异性、重复性及临床样本检出限进行评价。利用该方法对遵义地区273份未明确分型的肠道病毒阳性样本进行检测,将检出的部分CVA5阳性产物进行测序。结果成功建立一种基于实时荧光RT-PCR的CVA5检测方法,该方法在10^(4)~10^(9)copy/mL范围内具有良好的线性关系(R 2>0.999),平均扩增效率为102.26%。灵敏度高,对临床样本的检出限为103copy/mL。该方法的特异性强,与柯萨奇病毒A组2型(CVA2)、柯萨奇病毒A组4型(CVA4)、柯萨奇病毒A组6型(CVA6)、丙型肝炎病毒、呼吸道合胞病毒和鼻病毒等均不发生交叉反应。重复性实验的变异系数低于1.5%。273份未明确分型肠道病毒阳性样本中共检出25份CVA5,占未明确分型阳性样本的9.16%。结论该研究建立的一步法CVA5RT-PCR检测方法灵敏度高、特异性强、重复性好,可用于CVA5的检测。

RT-PCR技术在新型冠状病毒核酸检测的应用及质量评价分析

在新型冠状病毒感染患者的诊断中,采取有效检测方式进行及早诊断尤为重要。逆转录-聚合酶链式反应(RT-PCR)技术有较高灵敏度和特异度。本文分析临床应用RT-PCR技术检测新型冠状病毒的常见问题和应对策略,旨在为疫情防控常态化形势下临床实验室正确、高效开展新型冠状病毒检测提供参考意见。

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats

AIM:To investigate the stability of the seven housekeeping genes:beta-actin(ActB),glyceraldehyde-3-phosphate dehydrogenase(GAPDH),18s ribosomal unit 5(18s),cyclophilin A(CycA),hypoxanthine-guanine phosphoribosyl transferase(HPRT),ribosomal protein large P0(36B4)and terminal uridylyl transferase 1(U6)in the diabetic retinal tissue of rat model.METHODS:The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR)in two groups;normal control rats and streptozotocininduced diabetic rats.The stability analysis of gene expression was investigated using geNorm,NormFinder,BestKeeper,and comparative delta-Ct(ΔCt)algorithms.RESULTS:The 36B4 gene was stably expressed in the retinal tissues of normal control animals;however,it was less stable in diabetic retinas.The 18s gene was expressed consistently in both normal control and diabetic rats’retinal tissue.That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats.Furthermore,there was no ideal gene stably expressed for use in all experimental settings.CONCLUSION:Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Quantification of mRNA Levels by Fluorescently Labelled Reverse Transcription Competitive PCR

A reproducible,quantitative,non-radioactive method for the analysis of mRNA expression is described.After RNA preparation and cDNA synthesis,the cDNA was co-amplified with an internal standard in the same PCR system.The PCR products containing both targen and internal standard amplificates were electrophoresed and detected on an ABI 377 DNA Sequencer.For each sample,β-actin was also quantified by an identical procedure to compensate for relative differences between samples in the integrity of the individual RNA samples and for variations in reverse transcription.Due to the linear relationship between cDNA content and PCR product ratio of target cDNA template and competitive standard,a single PCR reaction was sufficient for quantification of a sample.The experimental results showed that the method is a mRNA quantitative RT-PCR method with high sensitivity and good reproducibility.It can be used in large-scale accurate quantitative analyses of mRNA expression of any gene.

逆转录-聚合酶链反应(RT-PCR)技术同时检测水中多种肠道病毒

建立了一种利用通用引物RT-PCR技术检测水中肠道病毒的方法.利用脊髓灰质炎病毒1～3型,柯萨奇病毒B3型作为参考病毒株,根据肠道病毒RNA5′非编码区中具有高度同源性的序列来设计通用引物.比较了M-MLV酶和AMV酶的逆转录效果,AMV酶能够成功地从地表水和生活污水中逆转录病毒RNA,更适于实际应用.对比研究了PCR过程中的退火温度,c(Mg2+)等因素对RT-PCR检测结果的影响,选择退火温度55℃,c(Mg2+)为2 mmol/L的反应条件,优化了RT-PCR检测方法.通过检测水样中接种的连续稀释的病毒,确定了该检测方法的灵敏度为38 CCID50.考察人工污染的地表水、污水、二级处理出水样品发现,检测灵敏度基本一致.该方法可应用在实际环境的肠道病毒检测中.

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 6 2 2 %。从百色、柳州地区等采集的健康猪扁桃体和淋巴结共 2 76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 ,2 4 6份扁桃体中有 35份阳性 ,占 14 2 %。采自柳州健康猪的 2 6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快速诊断禽流感

根据禽流感病毒ＮＰ基因的序列分析结果，设计了一对ＮＰ基因特异的引物。采用该对引物，不经病毒分离，直接从禽流感病毒感染鸡的气管、泄殖腔棉拭子和组织样品中提取核酸， ＲＴ～ＰＣＲ可以扩增出 ３２６ｂｐ的 ＮＰ基因片段。采用该技术对１４个亚型禽流感病毒标准参考株，４个亚型１２株国内分离野毒株，ＲＴ－ＰＣＲ检测的结果都呈阳性；对新城疫病毒、传染性法氏囊病毒、传染性支气管炎病毒、传染性喉气管炎病毒以及减蛋综合症病毒，ＲＴ－ＰＣＲ扩增结果都呈阴性。禽流感病毒 Ａ／Ｇｏｏｓｅ／Ｇｕａｎｇｄｏｎｇ（Ｈ５Ｎ１）和 Ａ／Ａｆｒｉｃａｎ Ｓｔａｒｌｉｎｇ／Ｅｎｇｌａｎｄ（Ｈ７Ｎ１）实验感染鸡样品 ＲＴ－ＰＣＲ检测与鸡胚病毒分离阳性率分别为３４／４２、３２／４２； ２４／５５、２４／５５， 二者符合率大于９５％。 ＲＴ－ＰＣＲ最少可检测到１０ｐｇ的病毒核酸。对山东某地发病鸡场样品进行ＲＴ－ＰＣＲ检测，只用６个小时就可得出准确的诊断结果，证明ＲＴ－ＰＣＲ检测方法敏感特异，可用于禽流感的快速诊断。

RT-PCR检测猪瘟病毒初探

本研究建立了反转录聚合酶链反应 ( RT PCR)技术检测猪瘟病毒 ( Hog Cholera Virus,HCV)方法。合成的二对引物 HCV 1 / HCV 2和 HCV A1 / HCV A2成功地扩增兔化弱毒株、法国株和石门株的核酸 ,并对采自博白县 3个、贵港市 1个和武宣县 1个猪场共 1 3份病料进行检测。结果从博白县和贵港市的 1 1份材料中检测到 HCV的核酸。

实时定量RT-PCR的原理及方法

实时定量RT-PCR广泛应用于定量检测mRAN表达水平,为基础研究、分子药物学和生物技术研究提供了一种有力的方法。该法具有易操作、高通量、敏感性高和特异性强的特点,随着新酶、新探针和新仪器的发展而得到快速的发展。本文将对实时定量RT-PCR的定量原理、仪器应用、探针种类的研究进展以及细胞因子mRNA表达水平检测上的应用作一综述。