兩種傳染病病原體復(fù)合探針熒光PCR檢測(cè)方法的建立
發(fā)布時(shí)間：2019-05-27 09:41
【摘要】：目的:感染性疾病是人類進(jìn)入21世紀(jì)以來,對(duì)人類健康安全危害極大的疾患。它具有傳播速度快、感染性強(qiáng)、危害大的特點(diǎn)。經(jīng)呼吸道傳播和蚊蟲叮咬傳播是兩種主要的傳播途徑。如曾引起世界范圍內(nèi)關(guān)注的傳染性非典型肺炎(SARS)、人禽流感、甲型H1N1流感以及黃熱病等。因此,對(duì)傳染性致病病毒進(jìn)行快速準(zhǔn)確的診斷是保障公眾健康和防控疫情的關(guān)鍵之一。然而,現(xiàn)在大部分臨床衛(wèi)生醫(yī)療機(jī)構(gòu)使用的傳統(tǒng)微生物檢驗(yàn)法涉及培養(yǎng)、分離等步驟,操作繁瑣、檢測(cè)周期長(zhǎng)。本研究的目的是建立一種檢測(cè)經(jīng)呼吸道傳播和蚊蟲叮咬傳播兩種病毒的快速、特異的診斷方法(PCR熒光探針法)。避免一般PCR易出現(xiàn)假陽(yáng)性、交叉污染等問題,從而能對(duì)兩種病毒進(jìn)行快速又準(zhǔn)確的檢測(cè),進(jìn)而更好地指導(dǎo)臨床治療。方法:首先,閱讀有關(guān)人類副流感病毒和寨卡病毒的相關(guān)文獻(xiàn),確定本研究要檢測(cè)的特異基因,以此為依據(jù)設(shè)計(jì)引物和復(fù)合探針,然后以臨床樣本為模板制備質(zhì)粒參考品。其次,通過優(yōu)化PCR反應(yīng)體系和反應(yīng)條件來確定引物探針的最佳濃度和比例。最后,評(píng)價(jià)該檢測(cè)方法的靈敏性、重復(fù)性和特異性;選擇樣本核酸提取效率最高的試劑;模板的最佳用量;不同PCR儀器對(duì)檢測(cè)結(jié)果的影響以及臨床樣本的檢測(cè)。結(jié)果:查閱文獻(xiàn)后確定人類副流感病毒的特異基因?yàn)镠N基因。確定了PCR反應(yīng)的最佳反應(yīng)體系和反應(yīng)條件,其中HPIV1和HPIV3的上、下游引物在體系中的終濃度為0.4μmol/L,探針在體系中的終濃度為0.2μmol/L;HPIV2和內(nèi)標(biāo)的上、下游引物在體系中的終濃度為0.15μmol/L,探針的終濃度為0.06μmol/L;其HPIV1、HPIV2和HPIV3探針的熒光探針與淬滅探針的比例為F∶Q=1∶2。退火溫度為58℃,退火時(shí)間為30s。最終該試劑盒的各項(xiàng)性能指標(biāo)顯示該試劑盒最低可檢出不低于5×102 copies/μl濃度的樣本,重復(fù)檢測(cè)10次結(jié)果表明其CV值小于5%,對(duì)10個(gè)種屬相近的病原體檢測(cè)并沒有擴(kuò)增,說明該試劑盒特異性良好。且兩種樣本核酸提取試劑效果相當(dāng),樣本用量選取為3μl。三種不同機(jī)型檢測(cè)結(jié)果表明不同機(jī)型對(duì)實(shí)驗(yàn)結(jié)果影響不大。臨床樣本檢測(cè)結(jié)果符合率為100%。確定寨卡病毒的特異基因?yàn)镹S5基因。引物探針性能評(píng)價(jià)結(jié)果表明其符合引物探針設(shè)計(jì)原則。檢測(cè)靈敏度可達(dá)到5×102 copies/μl,重復(fù)檢測(cè)10次結(jié)果表明其CV值小于10%。建立的檢測(cè)方法與其他種屬無交叉。說明本試劑盒具有良好的靈敏性、精密性和特異性。結(jié)論:本研究成功建立了一種可同時(shí)檢測(cè)人類副流感病毒3個(gè)亞型和寨卡病毒的診斷方法(PCR熒光探針法)。該方法靈敏度高、重復(fù)性好、特異性良好。為人類副流感病毒和寨卡病毒提供了一種特異、安全、快速、準(zhǔn)確的檢測(cè)方法,可以用于疾病的早期診斷,以指導(dǎo)及時(shí)、準(zhǔn)確地采取防護(hù)和治療措施。
[Abstract]:Objective: infectious disease is a disease that is very harmful to human health and safety since the beginning of the 21 st century. It has the characteristics of fast transmission, strong infection and great harm. Respiratory tract transmission and mosquito bite transmission are the two main routes of transmission. Such as infectious atypical pneumonia (SARS), avian influenza, H1N1 influenza and yellow fever, which have attracted worldwide attention. Therefore, the rapid and accurate diagnosis of infectious pathogenic virus is one of the keys to ensure public health and prevent and control the epidemic situation. However, most of the traditional microbiological testing methods used in clinical health and medical institutions involve culture, separation and other steps, the operation is tedious and the detection cycle is long. The purpose of this study was to establish a rapid and specific diagnostic method for the detection of respiratory tract transmission and mosquito bite transmission (PCR fluorescence probe method). Avoid false positive, cross-contamination and other problems in general PCR, so that the two viruses can be detected quickly and accurately, and then better guide clinical treatment. Methods: firstly, the literature on human parainfluenza virus and Zika virus was read, the specific genes to be detected in this study were determined, and primers and composite probes were designed on the basis of which primers and composite probes were designed, and then the plasmid reference materials were prepared by using clinical samples as templates. Secondly, the optimum concentration and proportion of primer probe were determined by optimizing PCR reaction system and reaction conditions. Finally, the sensitivity, reproducibility and specificity of the detection method were evaluated, the reagent with the highest nucleic acid extraction efficiency was selected, the optimum dosage of template, the influence of different PCR instruments on the detection results and the detection of clinical samples were selected. Results: after consulting the literature, the specific gene of human parainfluenza virus was identified as HN gene. The optimum reaction system and reaction conditions of PCR reaction were determined, in which the final concentration of downstream primers in the system of HPIV1 and HPIV3 was 0.4 渭 mol / L, and the final concentration of probe in the system was 0.2 渭 mol / L. The final concentration of upstream and downstream primers in HPIV2 and internal standard was 0.15 渭 mol / L, and the final concentration of probe was 0.06 渭 mol / L, and the ratio of fluorescence probe to quenching probe of HPIV1,HPIV2 and HPIV3 probe was F 鈮，
本文編號(hào)：2486046
[Abstract]:Objective: infectious disease is a disease that is very harmful to human health and safety since the beginning of the 21 st century. It has the characteristics of fast transmission, strong infection and great harm. Respiratory tract transmission and mosquito bite transmission are the two main routes of transmission. Such as infectious atypical pneumonia (SARS), avian influenza, H1N1 influenza and yellow fever, which have attracted worldwide attention. Therefore, the rapid and accurate diagnosis of infectious pathogenic virus is one of the keys to ensure public health and prevent and control the epidemic situation. However, most of the traditional microbiological testing methods used in clinical health and medical institutions involve culture, separation and other steps, the operation is tedious and the detection cycle is long. The purpose of this study was to establish a rapid and specific diagnostic method for the detection of respiratory tract transmission and mosquito bite transmission (PCR fluorescence probe method). Avoid false positive, cross-contamination and other problems in general PCR, so that the two viruses can be detected quickly and accurately, and then better guide clinical treatment. Methods: firstly, the literature on human parainfluenza virus and Zika virus was read, the specific genes to be detected in this study were determined, and primers and composite probes were designed on the basis of which primers and composite probes were designed, and then the plasmid reference materials were prepared by using clinical samples as templates. Secondly, the optimum concentration and proportion of primer probe were determined by optimizing PCR reaction system and reaction conditions. Finally, the sensitivity, reproducibility and specificity of the detection method were evaluated, the reagent with the highest nucleic acid extraction efficiency was selected, the optimum dosage of template, the influence of different PCR instruments on the detection results and the detection of clinical samples were selected. Results: after consulting the literature, the specific gene of human parainfluenza virus was identified as HN gene. The optimum reaction system and reaction conditions of PCR reaction were determined, in which the final concentration of downstream primers in the system of HPIV1 and HPIV3 was 0.4 渭 mol / L, and the final concentration of probe in the system was 0.2 渭 mol / L. The final concentration of upstream and downstream primers in HPIV2 and internal standard was 0.15 渭 mol / L, and the final concentration of probe was 0.06 渭 mol / L, and the ratio of fluorescence probe to quenching probe of HPIV1,HPIV2 and HPIV3 probe was F 鈮，
本文編號(hào)：2486046
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