7.人乳头瘤病毒核酸分型检测-透景

7.人乳头瘤病毒核酸分型检测-透景 Shanghai Fenglin Clinical Laboratory Co.,Ltd. 上海枫林医药医学检验有限公司 Standard operation procedure for detection of nucleic acid of TITLE:File Code human papillomavirus with PCR-flow fluorescence assay 文件编号: SOP FL-SOP-CL-6007 文件名称:人乳头瘤病毒核酸分型检测 标准 excel标准偏差excel标准偏差函数exl标准差函数国标检验抽样标准表免费下载红头文件格式标准下载 操作规程 WRITTEN BY/ DATE REVIEW BY/ DATE APPROVED BY/ DATE 编写者: 罗孝伟 审阅者: 黄永富 批准者: 日 期 : 日 期 : 2012-07-20 日 期 : 2012-07-20 REVISION DATE New Format/Revision Number EFFECTIVE DATE: 生效日期: 修订日期: 2011-12-31 新版/修号: 2/0 REPLACE NUMBER 被替代版号: 1/0 1. Intended Use目的 通过病毒核酸扩增定性检测人体宫颈脱落细胞中人乳头瘤病毒分型。 2. Sphere of Application适用范围 人乳头瘤病毒分型定性检测。 3. Principle of Test检验方法及原理 采用多重PCR技术对检测样品的核酸DNA进行扩增，并用包被有核酸探针的多种编码微球 和扩增产物进行杂交，结果用流式阵点仪检测分析。流式荧光杂交法是将PCR扩增产物盒 微球液进行混和，并经过杂交、荧光素标记、Luminex流式分析仪阅读得到相应的检测结 果。微球杂交液中包含多种荧光编码微球，每种荧光荧光编码微球的表面都共价交联有与 亚型对应的寡核苷酸探针，在杂交过程中探针特异性识别并结合PCR产物中的靶序列，经 过和荧光素反应后形成微球-探针-扩增产物-荧光素的复合物，经Luminex流式分析仪阅读 时，可获得每种复合物的微球编码及其对应的荧光强弱信号，最后经过透景HPV专用的分 析软件判读相应的检测结果。 4. Specimen Collection使用的样本 4.1. 样本种类:阴道或尿道分泌物。 4.2. 收集方法:医护人员先以窥阴器或阴道张开器暴露宫颈，用棉拭子将宫颈口过多的分泌 物擦去。取出宫颈刷置于宫颈口，单方向旋转4-5周以获得足量的上皮细胞样本，然后将 宫颈刷头部放入洗脱管中，沿刷柄折痕处将宫颈刷柄折断，旋紧洗脱管盖，做好样本标 识，保持洗脱管直立放置。 rational concept to allow network more in line with the objective laws of the future development of the city. 1.5.2 planning research areas: throughout the city, with an area of 13,000 km2, cover 2 districts and 8 counties of the city, including downtown and city levels. Planning range: main for City Center City, area for 1268km2, North to cases field Xiang, and Jin Pingzhen, and Gao Dian Zhen, and huangshazhen, and Liu home town North Township territories, East Nan XI Zhen, and Jiangnan East Township territories, South to horse home, and song home, and Li Zhuang Zhen, and South wide town, and Zhao field Street do South administrative border, West to baixizhen, and dish dam town, and thought slope Xiang West Township territories. 1.6 planning target 1.6.1 General target this items research to will Yibin city built Yangtze River upstream chuan Yunnan Guizhou combined Department Regional Center City, support city space of ordered expand for target, reference Yibin city integrated traffic planning (2014-2030 years) and the Yibin City City general planning (2013-2030 years) of research results, using science, and advanced of network research method, building more level, and more mode of track traffic system, Meet at different levels of the city's rail transportation needs and quality requirements. 1.6.2 target 1, refined urban development axis of urban development concepts, judgments, rail-guided downtown from solo to group-development of the network, from one Center to center city with axial development. 2, network planning adapted to Yibin City status and future development needs, demonstrates rational scale of rail transport to meet the needs 4.3. 病人准备要求:月经正常妇女，在月经来潮后10-18天为最佳检查时间;检查前三天内不 要作阴道冲洗，不要用避孕药膏等阴道内用药物;检查前24小时不应有性行为;检查前 不进行醋酸或碘液涂抹。 4.4. 样本容器的要求:专用样本保存液的容器。 4.5. 样本用量:50-200μl，应保证有足够的量用于检测。 4.6. 拒收样本的规定:除阴道或尿道分泌物以外的样本，应予以拒收。 4.7. 样本的处理方法:高速冷冻离心机13000转/min离心5min备用。 4.8. 样本的储存规定: 冷藏 冷冻 冷冻 室温 复融 (2-8?) (-20?) (-70?以下) 新鲜样本 12小时 7天 3个月 长期保存 1次 样本的运输 采用冰壶加冰或泡沫箱加冰密封进行运输，在途时限不宜超过48小时 样本储存 应在检测完成7天内储存，不得超过7天 4.9. 样本的外送规定: 4.9.1. 仪器故障不能立即维修及试剂断货导致不能在 报告 软件系统测试报告下载sgs报告如何下载关于路面塌陷情况报告535n,sgs报告怎么下载竣工报告下载 周期内准时发出检测报告时，样本可 外送至其他有医疗资质的单位进行检测。 4.9.2. 样本结果可疑或与临床不符时，样本可外送至其他有医疗资质的单位进行复查比对。 5. Reagents，calibrators，quality control试剂、标准品、质控品 5.1. 试剂、标准品/校准品、质控品的产品名 5.1.1. 试剂:上海透景生命科技有限公司高危型人乳头瘤病毒核酸分型检测试剂盒(流式荧光 杂交法)。 5.1.2. 标准品/校准品:N/A 5.1.3. 质控品:上海透景生命科技有限公司。 5.2. 试剂、标准品/校准品、质控品的组成及包装规格 5.2.1. 主要试剂组成成分: guided downtown fro-ty requirements. 1.6.2 target 1, refined urban development axis of urban development concepts, judgments, railmore level, and more mode of track traffic system, Meet at different levels of the city's rail transportation needs and quali 2030 years) of research results, using science, and advanced of network research method, building-2030 years) and the Yibin City City general planning (2013-ort city space of ordered expand for target, reference Yibin city integrated traffic planning (2014supp items research to will Yibin city built Yangtze River upstream chuan Yunnan Guizhou combined Department Regional Center City, aixizhen, and dish dam town, and thought slope Xiang West Township territories. 1.6 planning target 1.6.1 General target thist to bhorse home, and song home, and Li Zhuang Zhen, and South wide town, and Zhao field Street do South administrative border, Wesangshazhen, and Liu home town North Township territories, East Nan XI Zhen, and Jiangnan East Township territories, South to and hulanning range: main for City Center City, area for 1268km2, North to cases field Xiang, and Jin Pingzhen, and Gao Dian Zhen, out the city, with an area of 13,000 km2, cover 2 districts and 8 counties of the city, including downtown and city levels. Presearch areas: throughrational concept to allow network more in line with the objective laws of the future development of the city. 1.5.2 planning status and future development needs, demonstrates rational scale of rail transport to meet the needsdevelopment of the network, from one Center to center city with axial development. 2, network planning adapted to Yibin City -m solo to group2 《人乳头瘤病毒核酸分型检测标准操作规程》 组成成分 数量 规格 核酸提取组份 核酸提权缓冲液 1瓶 12mL 引物混合液 2管 150uL PCR预混液 2管 300,L 核酸扩增组份 聚合酶 1管 48uL 质控品 1管 200uL 微球杂交液 1瓶 1.2mL 杂交组份 SA-PE 1管 4mL 5.2.2. 标准品/校准品:N/A 5.2.3. 质控品:200μl×1支。 5.3. 试剂、标准品/校准品、质控品稳定性与贮存 名称 储存条件 机载稳定性 准备 试剂 杂交组份于2-8?避光、N/A 冻存试剂室温融化并振荡 防冻保存;核酸提取组份混匀后，2000rpm离心10 于室温或2-8?保存;核秒钟。 酸扩增组份于-20?以下 冷冻存放;试剂盒有效期 为6个月。 标准品/校准品 N/A N/A N/A 质控品 -20?以下冷冻存放至有N/A 室温放置至少15分钟使 效期截止日期。 其充分复融，颠倒或旋涡 振荡混匀后使用。 6. Instrument仪器 6.1. 美国ABI7500 实时荧光定量PCR仪; 6.2. 新加坡ESCO Airstream A2型二级生物安全柜; 6.3. 上海培清科技有限公司JS-400A电热恒温金属浴; 6.4. Thermo CL31R低温高速离心机; 6.5. 美国Luminex200多功能流式点阵仪。 7. Procedure操作步骤 7.1. 扩增试剂准备(PCR前准备区) 按下表加入PCR试剂和样品: rational concept to allow network more in line with the objective laws of the future development of the city. 1.5.2 planning research areas: throughout the city, with an area of 13,000 km2, cover 2 districts and 8 counties of the city, including downtown and city levels. Planning range: main for City Center City, area for 1268km2, North to cases field Xiang, and Jin Pingzhen, and Gao Dian Zhen, and huangshazhen, and Liu home town North Township territories, East Nan XI Zhen, and Jiangnan East Township territories, South to horse home, and song home, and Li Zhuang Zhen, and South wide town, and Zhao field Street do South administrative border, West to baixizhen, and dish dam town, and thought slope Xiang West Township territories. 1.6 planning target 1.6.1 General target this items research to will Yibin city built Yangtze River upstream chuan Yunnan Guizhou combined Department Regional Center City, support city space of ordered expand for target, reference Yibin city integrated traffic planning (2014-2030 years) and the Yibin City City general planning (2013-2030 years) of research results, using science, and advanced of network research method, building more level, and more mode of track traffic system, Meet at different levels of the city's rail transportation needs and quality requirements. 1.6.2 target 1, refined urban development axis of urban development concepts, judgments, rail-guided downtown from solo to group-development of the network, from one Center to center city with axial development. 2, network planning adapted to Yibin City status and future development needs, demonstrates rational scale of rail transport to meet the needs 第3 页 共7 页 试 剂 用 量(单人份) 引物混合液 5µl PCR预混液 10µl Taq 0.8µl 模板DNA 5µl 计算好各试剂的使用量，加入适当体积离心管中，充分混合均匀，2000rpm离心10秒，向 设定的n个PCR 反应管中分别加入15μl，转移至样本处理区。 7.2. 样本处理(样本处理区) 7.2.1 轻微振荡悬浮起保存液中的脱落细胞，确保细胞均匀悬浮起来。 7.2.2 取50μL保存液体于1.5 ml 离心管中，如果有颗粒状物体(疣体)确保有一部分取入离 心管。 7.2.3 12000rpm 3分钟， 小心取上清并丢弃。 7.2.4 加入200µl标本提取液，振荡混匀，将离心管放入100ºC金属浴10min。 7.2.5 12000 rpm离心5分钟。保留上清以备检测。 7.3. 加样 向所设定的n个PCR反应管中分别加入步骤7.2中处理过的样本5μl，盖紧管盖，将PCR反应管转移至传递窗，记录样本摆放顺序。 7.4. PCR扩增(检测区) 7.4.1. PCR仪器循环条件设置: 95? 3min 95? 30s 58? 30s 5cycles 72? 30s 95? 30s 55? 30s 35cycles 72? 30s 72? 3min 7.4.2. 根据需要反应的数量裁剪反应板，在每孔中加入微球杂交液22 µl。 7.4.3. 在每孔中加入PCR产物3µl，依次按顺序加样，加样顺序和扩增时的顺序保持一致，避 免加样错误。 -ty requirements. 1.6.2 target 1, refined urban development axis of urban development concepts, judgments, railmore level, and more mode of track traffic system, Meet at different levels of the city's rail transportation needs and quali 2030 years) of research results, using science, and advanced of network research method, building-2030 years) and the Yibin City City general planning (2013-ort city space of ordered expand for target, reference Yibin city integrated traffic planning (2014supp items research to will Yibin city built Yangtze River upstream chuan Yunnan Guizhou combined Department Regional Center City, aixizhen, and dish dam town, and thought slope Xiang West Township territories. 1.6 planning target 1.6.1 General target thist to bhorse home, and song home, and Li Zhuang Zhen, and South wide town, and Zhao field Street do South administrative border, Wesangshazhen, and Liu home town North Township territories, East Nan XI Zhen, and Jiangnan East Township territories, South to and hulanning range: main for City Center City, area for 1268km2, North to cases field Xiang, and Jin Pingzhen, and Gao Dian Zhen, out the city, with an area of 13,000 km2, cover 2 districts and 8 counties of the city, including downtown and city levels. Presearch areas: throughrational concept to allow network more in line with the objective laws of the future development of the city. 1.5.2 planning status and future development needs, demonstrates rational scale of rail transport to meet the needsdevelopment of the network, from one Center to center city with axial development. 2, network planning adapted to Yibin City -m solo to groupguided downtown fro4 《人乳头瘤病毒核酸分型检测标准操作规程》 7.4.4. 剪下合适的封口膜，封住反应板，放入PCR仪中，运行杂交程序。 7.4.5. 先95?变性5分钟，然后48?杂交30分钟后在PCR仪上撕去封口膜(不要将反应板从PCR 仪上拿下来)。 7.4.6. 每孔加入75µl SA-PE，重新用封口膜封住反应板。 7.4.7. 提前打开Luminex 200的加热功能，48?孵育15分钟，上机检测。Luminex的操作见 “Luminex 200 操作、维护、校准标准手册”。 7.5. 质量控制 运行质控: 记录室内质控结果。 每工作日应于病人样本检测同时运行进行两种质控品的检测，如果质控结果未满足以下接受质控频率: 标准，样本必须重测。 阴性内对照微球上包被的是与HPV和Globin无关的一段核酸序列，信号值应与本底相当， 否则实验判为无效。 质控结果接受阳性内对照微球上包被的是人基因组蛋白的探针，PCR组份中包含能扩增Globin的引物系标准 统，出现相应亚型的信号值，否则判为无效。 1、 确认试剂及质控品在有效期内，并正确使用。 质控失控解决2、 在辨别和改正造成背离的原因之前不可发布病人检测结果。 方案 气瓶 现场处置方案 .pdf气瓶 现场处置方案 .doc见习基地管理方案.doc关于群访事件的化解方案建筑工地扬尘治理专项方案下载 3、 必要时请联系工程师。 8. Precautions and Warnings操作注意事项 8.1. 所有试剂必须完全融解后才能使用。 8.2. 每次配置PCR试剂时需要多配置1-3人份以抵消移液器的误差及移液耗损。 8.3. 加杂交微球时，应充分混匀。 8.4. 加样时要严格操作避免污染。 8.5. 加样完成后10000转离心3秒，以使管壁上的液体聚集到管底并彻底赶走反应体系内的 气泡。 8.6. 样本处理必须做好安全防护 措施 《全国民用建筑工程设计技术措施》规划•建筑•景观全国民用建筑工程设计技术措施》规划•建筑•景观软件质量保证措施下载工地伤害及预防措施下载关于贯彻落实的具体措施 ，防止感染。 8.7. 每天或在每次检测完成后，各操作室的工作台面需用可移动紫外灯(近工作台面)照射 30分钟以上，以防止扩增产物对下次检测造成污染。 8.8. 检测过的样品及其它实验废弃物应置于医疗垃圾回收袋统一处理。 9. Calculations计算方法 仪器自行检测并计算。 10. Reference Range参考范围 rational concept to allow network more in line with the objective laws of the future development of the city. 1.5.2 planning research areas: throughout the city, with an area of 13,000 km2, cover 2 districts and 8 counties of the city, including downtown and city levels. Planning range: main for City Center City, area for 1268km2, North to cases field Xiang, and Jin Pingzhen, and Gao Dian Zhen, and huangshazhen, and Liu home town North Township territories, East Nan XI Zhen, and Jiangnan East Township territories, South to horse home, and song home, and Li Zhuang Zhen, and South wide town, and Zhao field Street do South administrative border, West to baixizhen, and dish dam town, and thought slope Xiang West Township territories. 1.6 planning target 1.6.1 General target this items research to will Yibin city built Yangtze River upstream chuan Yunnan Guizhou combined Department Regional Center City, support city space of ordered expand for target, reference Yibin city integrated traffic planning (2014-2030 years) and the Yibin City City general planning (2013-2030 years) of research results, using science, and advanced of network research method, building more level, and more mode of track traffic system, Meet at different levels of the city's rail transportation needs and quality requirements. 1.6.2 target 1, refined urban development axis of urban development concepts, judgments, rail-guided downtown from solo to group-development of the network, from one Center to center city with axial development. 2, network planning adapted to Yibin City status and future development needs, demonstrates rational scale of rail transport to meet the needs 第5 页 共7 页 仪器自行检测并计算。 11. Operating Performance操作性能 11.1. 灵敏度:本试剂盒的最低检测限为10-20拷贝(0.02ng/ml)。 11.2. 特异性:阳性参比拼品符合率:试剂盒对26种HPV亚型的阳性参比品进行2次平行检测， 结果均为阳性; 阴性参比品符合率:试剂盒对正常人基因组参比品进行2次平行检测，结果均为阴性。 11.3. 重复性:分别对HPV16、HPV18、HPV31、HPV33和HPV45共5种高危亚型阳性参比品进行 10次平行检测，结果一致且准确。 12. Reporting Results and Interpretation结果报告及解释 12.1. 将20余种探针信号的中位数作为计算的背景信号。 12.2. 如果阳性内对照Globin的信号大于150，且大于等于2.5倍背景信号值，即代表整个实 验成功。如果不符合前述条件，代表实验失败。可能的原因是样本中没有采集到细胞、 某种试剂失效或者相关仪器设备故障。 12.3. 如果有任何亚型探针的信号大于150，且大于等于2.5倍背景信号值，即判断为该探针对 应的亚型阳性。如果不符合前述条件的，即判断为阴性。 13. Limitations of Procedure方法局限性 18、31、33、35、39、45、51、52、56、58、59和66共1313.1. 可检测WHO确认的HPV 16、 种高危亚型，此外还可以检测其他6种高危亚型(HPV26、53、55、68、82、83)和7种 低位亚型(HPV6、11、40、42、44、61、73)，对于其它亚型感染本品不能检出。 13.2. 无法检测病毒载量为10-20copies/mL以下的样本。 14. Clinical Significance临床意义 人类乳头瘤病毒(HPV)可以通过多种途径感染生殖道，从而导致尖锐湿疣以及宫颈病变， 甚至可能引起宫颈癌的发生。从高危型HPV的持续感染到一般的宫颈癌前病变并最终发展为 宫颈癌大约需要5-10年。因此，有针对性地进行HPV的分型检测对于宫颈癌的早期诊断和 治疗具有一定的意义。 本方法可以对人乳头瘤病毒 (HPV) 的感染情况进行定性检测并加以分型，能够: 1) 检测26种HPV亚型，直接提示感染HPV的基因亚型; 2) 包括19种高危亚型:HPV116、18、31、33、35、39、45、51、52、56、58、55、66、 26、53、55、68、82、83;7种低危亚型:HPV 6、11、40、42、44、61、73; 3) 可用于临床HPV感染的辅助诊断。 本方法只能检测是否有HPV感染，但不能确定是否已经发生病变或者病变发展到何种阶段。 2030 years) of research results, using science, and advanced of network research method, building-2030 years) and the Yibin City City general planning (2013-ort city space of ordered expand for target, reference Yibin city integrated traffic planning (2014supp items research to will Yibin city built Yangtze River upstream chuan Yunnan Guizhou combined Department Regional Center City, aixizhen, and dish dam town, and thought slope Xiang West Township territories. 1.6 planning target 1.6.1 General target thist to bhorse home, and song home, and Li Zhuang Zhen, and South wide town, and Zhao field Street do South administrative border, Wesangshazhen, and Liu home town North Township territories, East Nan XI Zhen, and Jiangnan East Township territories, South to and hulanning range: main for City Center City, area for 1268km2, North to cases field Xiang, and Jin Pingzhen, and Gao Dian Zhen, out the city, with an area of 13,000 km2, cover 2 districts and 8 counties of the city, including downtown and city levels. Presearch areas: throughrational concept to allow network more in line with the objective laws of the future development of the city. 1.5.2 planning status and future development needs, demonstrates rational scale of rail transport to meet the needsdevelopment of the network, from one Center to center city with axial development. 2, network planning adapted to Yibin City -m solo to groupguided downtown fro-ty requirements. 1.6.2 target 1, refined urban development axis of urban development concepts, judgments, railmore level, and more mode of track traffic system, Meet at different levels of the city's rail transportation needs and quali 6 《人乳头瘤病毒核酸分型检测标准操作规程》 若检测结果为阳性，建议进一步做细胞学检测。 15. References参考文献 15.1. 上海透景生命科技有限公司高危型人乳头瘤病毒核酸分型检测试剂盒 说明书 房屋状态说明书下载罗氏说明书下载焊机说明书下载罗氏说明书下载GGD说明书下载 。 15.2. 叶应妩，王毓三，申子瑜主编)全国临床检验操作规程)第三版)南京:东南大学出版 社，2006。 Revision History 修订历史记录页 版本号 修订摘要 编写者 2/0 新修订。 罗孝伟 Document Annually Reviewing 文件年度审核记录 REVIEW BY/ DATE REVIEW BY/ DATE REVIEW BY/ DATE 审阅者: 审阅者: 审阅者: 日 期 : 日 期 : 日 期 : REVIEW BY/ DATE REVIEW BY/ DATE REVIEW BY/ DATE 审阅者: 审阅者: 审阅者: 日 期 : 日 期 : 日 期 : rational concept to allow network more in line with the objective laws of the future development of the city. 1.5.2 planning research areas: throughout the city, with an area of 13,000 km2, cover 2 districts and 8 counties of the city, including downtown and city levels. Planning range: main for City Center City, area for 1268km2, North to cases field Xiang, and Jin Pingzhen, and Gao Dian Zhen, and huangshazhen, and Liu home town North Township territories, East Nan XI Zhen, and Jiangnan East Township territories, South to horse home, and song home, and Li Zhuang Zhen, and South wide town, and Zhao field Street do South administrative border, West to baixizhen, and dish dam town, and thought slope Xiang West Township territories. 1.6 planning target 1.6.1 General target this items research to will Yibin city built Yangtze River upstream chuan Yunnan Guizhou combined Department Regional Center City, support city space of ordered expand for target, reference Yibin city integrated traffic planning (2014-2030 years) and the Yibin City City general planning (2013-2030 years) of research results, using science, and advanced of network research method, building more level, and more mode of track traffic system, Meet at different levels of the city's rail transportation needs and quality requirements. 1.6.2 target 1, refined urban development axis of urban development concepts, judgments, rail-guided downtown from solo to group-development of the network, from one Center to center city with axial development. 2, network planning adapted to Yibin City status and future development needs, demonstrates rational scale of rail transport to meet the needs 第7 页 共7 页