CN116042929B - 检测引起发热出疹性疾病病毒的组合物及其应用 - Google Patents
检测引起发热出疹性疾病病毒的组合物及其应用 Download PDFInfo
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Abstract
本发明属于医学检测领域,具体涉及检测引起发热出疹性疾病病毒的组合物。本发明的检测引起发热出疹性疾病病毒的组合物,包括用于检测猴痘病毒、天花病毒、单纯疱疹病毒I型、水痘‑带状疱疹病毒的引物和探针,其可检测上述四中病毒,外加一个人源内参基因,采用5色荧光PCR技术,设计为单管多通道检测产品。从而,本发明使用5色荧光技术,实现检测4种常见引物出疹疾病的病原体微生物,具体包括猴痘病毒、天花病毒、单纯疱疹病毒1型和水痘‑带状疱疹病毒,同时使用人源基因GAPDH作为内对照,对标本采集、运输、保存、提取和加样等全过程进行质量监控,具有特异性好、便于运输和保存的特点。
Description
技术领域
本发明属于医学检测领域,具体涉及检测引起发热出疹性疾病病毒的组合物。
背景技术
皮疹常见于传染性疾病,如麻疹、猩红热、水痘、伤寒及流行性脑脊髓膜炎等。临床引起皮疹的疾病颇多。鉴别诊断通常包括天花、水痘、麻疹、细菌性皮肤感染、疥疮、药物过敏和梅毒。
猴痘是由猴痘病毒引起的一种罕见、散发的、天花样临床表现的急性传染病。在人类中,猴痘是一种散发性偶见疾病,其体征和症状类似天花,但病情通常较轻。猴痘感染者有动物接触史,临床表现最初表现类似“流感”的症状,随后皮肤出现疱疹、经历脓疱、结痂后留有瘢痕。
单纯疱疹病毒I型(HSV I型)原发感染常局限在口咽部,病毒通过呼吸道或直接接触患者唾液传播,引起口龈炎,表现为发热、咽喉痛,在牙龈和咽颊部出现成群疱疹,疱疹破溃后形成溃疡,大量病毒集中在病灶内,此外,病毒还可引起疱疹性角膜结膜炎、唇疱疹、疱疹性脑炎和皮肤疱疹性湿疹等疾病。
水痘-带状疱疹病毒是指在儿童初次感染引起水痘,恢复后病毒潜伏在体内,少数病人在成人后病毒再发而引起带状疱疹,故被称为水痘-带状疱疹病毒。初期局部皮肤有异常感,瘙痒、疼痛,进而出现红疹、疱疹,串连成带状,以躯干和面额部为多见,呈单侧分布,病程约3周左右,少数可达数月之久。
天花是由天花病毒引起的一种烈性传染病,也是在世界范围内被人类消灭的一种传染病。感染天花病毒后的潜伏期平均约为12天(7-17天)。发病早期出现发热、寒战、头痛、嗜睡、乏力、背部疼痛和肌痛等前驱症状。发病1-3天后出现皮疹。首先出现再面部,逐渐蔓延至四周,手心和脚掌均可出现皮疹。
由于在1980年消灭了天花并随后停止接种天花疫苗,猴痘已成为公共卫生领域最重要的正痘病毒。2022年5月初以来,已有20多个非地方性流行国家发现多例猴痘病例,且已出现人际传播。
按照中国疾控中心发布的《猴痘防控技术指南(2022 版》内容,诊断猴痘疾病时需要与其他发热出疹性疾病,如水痘、带状疱疹、单纯疱疹、麻疹、登革热等进行鉴别诊断,实现疑似病例早发现、早报告、早诊断、早调查、早处置。
国际上尚无一种通用的、安全快速的对天花和猴痘病毒感染的血清学诊断方法,而生物学鉴别诊断(病毒分离培养、电镜观察等)通常需要3-6d,且存在于生物安全危害隐患。而血清学诊断于临床诊断的灵敏度不高,不易诊断天花、猴痘及其他出皮疹的疾病的问题。
发明内容
针对现有技术中存在的问题,并根据猴痘病毒快速检测和鉴别的需求,提供一种检测引起发热出疹性疾病病毒的组合物,采用TaqMan-MGB探针法,利用5种荧光标记,结合MGB探针,可实现单管五重检PCR扩增,检测猴痘病毒、天花病毒、单纯疱疹病毒I型(HSV I型)、水痘-带状疱疹病毒及人基因组基因,具有检测通量高,技术成熟、成本低、操作简单、结果判读简单易懂的特点。
根据本发明具体检测引起发热出疹性疾病病毒组合物,所述组合物包括用于检测猴痘病毒、天花病毒、单纯疱疹病毒I型、水痘-带状疱疹病毒的引物和探针,其中,
检测猴痘病毒的引物、探针为:
SEQID NO.1:5'-TAGAAAATGGAGCAAATGTCA-3';
SEQID NO.2:5'-CTACGGGCGATGACAGAT-3';
SEQID NO.3:5'-ACGCAGTAATGAGTAA-3';
检测天花病毒的引物、探针为:
SEQID NO.4:5'-AATATTATGAATGAGAGATACCTTAATGT-3';
SEQID NO.5:5'-ACATTTTTTATCTGTGAAAACAGG-3';
SEQID NO.6:5'-TGTGTCAAAAGTATACGA-3';
检测水痘-带状疱疹病毒株的引物、探针为:
SEQID NO.7:5'-GAGATTCACGAAGATTGCGG-3';
SEQID NO.8:5'-CCTCCCAGCACGCTAACG-3';
SEQID NO.9:5'-CGACTTTTATCTAATGGG-3';
检测天单纯疱疹病毒I型的引物、探针为:
SEQID NO.10:5'-GTCTGGTGGTCGTAGGGTG-3';
SEQID NO.11:5'-ATCTTGTGGGCGGGACT-3';
SEQID NO.12:5'-TGTATCTGGCGTTCG-3'。
根据本发明具体实施方式的组合物,其还包括人源内参基因GAPDH的引物、探针,
SEQID NO.13:5'-ATGGACACGCTCCCCTGAC-3';
SEQID NO.14:5'-TGAGTCCTTCCACGATACCAAA-3';
SEQID NO.15:5'-GCAATGCCTCCTGCACCACCAA-3'。
根据本发明具体实施方式的组合物,所述组合物还包括PCR反应液,扩增酶混合液、阳性对照和阴性对照。
优选的,所述组合物还包括10×PCR Buffer、 MgCl2、 dUTP/dNTPs Mix、所述引物和探针、BSA、Taq DNA聚合酶、UDG酶和灭菌超纯水。
根据本方具体实施方式的组合物,其中,所述探针的5’端标记荧光基团,3’端标记淬灭基团。
优选的,所述荧光基团包括FAM、VIC、ROX、CY5和CY5.5,并且检测猴痘病毒、天花病毒、单纯疱疹病毒I型、水痘-带状疱疹病毒的探针所标记的荧光基团彼此互不相同且互不干扰。
本发明还提供上述组合物在制备检测引起发热出疹性疾病病毒的试剂中的应用,适用的临床标本类型为血清、血液标本、咽拭子、疱疹液、疱疹表面或渗出液拭子等。
本发明的有益效果:
本发明使用5色荧光技术,检测4种常见引物出疹疾病的病原体微生物,具体包括猴痘病毒、天花病毒、单纯疱疹病毒1型和水痘-带状疱疹病毒,同时使用人源基因GAPDH作为内对照,对标本采集、运输、保存、提取和加样等全过程进行质量监控,具有特异性好、便于运输和保存的特点。
本发明的组合物可用于上述病原微生物的同时检测或鉴定,为临床判断发热出疹性疾病提供辅助指导。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
材料和试剂
1. 含有靶基因片段的假病毒参考品:自制;
2. 特异性引物探针:通用生物系统(安徽)有限公司;
3. 10×PCR Buffer(Mg2+free):宝日医生物技术(北京)有限公司,货号为9151AM,25mM MgCl2为10×PCR Buffer配套产品。dNTP/dUTP Mix:生工生物工程(上海)股份有限公司,货号为B300578;
4. 扩增酶混合物:Taq DNA聚合酶(产品规格为5 U/μl):生工生物工程(上海)股份有限公司,货号为B500010。UDG酶(产品规格为2U/μl):生工生物工程(上海)股份有限公司,货号为B110042;
5.DEPC处理水;自制;
6. 核酸提取试剂:自产,型号Ex-DNA/RNA;
7. 10份健康志愿者咽拭子:公司员工;
其他病原体阳性标本或培养物:单纯疱疹病毒-2标本、肠道病毒临床标本、灭活金黄色葡萄球菌培养物、灭活化脓性链球菌培养物、灭活绿脓杆菌培养物、灭活白色念珠菌培养物,医院惠赠。
实施例1引物探针的设计
目前文献报道,核酸检测天花病毒和猴痘病毒的靶序列都位于基因组的中心区域,本发明发现将靶序列设定于上述中心区域,产生非特异性反应,导致核酸检测结果不准确。
本发明选择基因组编码区末端的变异区作为检测靶序列,可有效避免天花病毒与猴痘病毒之间的非特异反应。
确定各病原体特异靶基因标志物,针对性的设计引物探针,其中探针5’端标记荧光基团,3’端标记淬灭基团。具体序列如下:
猴痘病毒株MPV-ZAI D1L(NC003310):c6110-4797
ATGGAGTCGGCAGATTTCATGGCTGTCGATGAGCAGTTTCACGACGACCTCGATCTTTGGTCATTATCTTTGGTAGATGACTATAAAAAACATGGATTAGGTGTTGACTGTTATGTTCTAGAACCAGTTGTTGACAGGAAAATATTTGATAGATTTCTCCTTGAACCAATTTGTGATCCTGTAGATGTTCTGTATGATTATTTTAGGATTCATAGAGATAATATTGATCAGTATATAGTAGATAGACTGTTTGCATATATTACATATAAAGATATTATATCTGCATTAGTGTCAAAGAATTATATGGAAGATATTTTCTCTATAATTATTAAGAATTGTAATTCTGTACAAGATCTCTTACTTTACTATCTATCTAATGCATATGTAGAAATAGACATTGTTGATTTTATGGTAGATCATGGGGCTGTAATATATAAAATAGAATGCTTGAATGCCTATTTTAGGGGAATATGTAAAAAGGAAAGTAGTGTTGTTGAGTTTATTTTGAATTGTGGTATCCCAGATGAAAATGATGTTAAATTAGATCTATATAAAATAATTCAGTATACTAGGGGATTCCTTGTAGATGAACCCACAGTATTAGAAATTTATAAGCTTTGTATCCCATATATTGAAGATATCAATCAACTAGATGCTGGTGGAAGGACCTTGCTTTATCGCGCTATCTATGCAGGTTATATAGATTTAGTATCATGGCTATTAGAAAATGGAGCAAATGTCAACGCAGTAATGAGTAATGGATATACATGTCTTGACGTGGCCGTGGATAGGGGATCTGTCATCGCCCGTAGGGAAACACATCTTAAAATATTAGAAATATTGCTTAGAGAACCATTGTCTATTGACTGTATAAAATTAGCTATACTTAATAATACAATTGAAAACCATGATGTGATAAAGCTCTGTATCAAGTATTTTATGATGGTAGATTATTCACTTTGTAATGTGTATGCATCATCACTCTTTGATTATATAATTGATTGTAAACAAGAATTGGAGTACATTAGGCAGATGAAAATTCATAATACAACCATGTATGAGTTAATCTATAATAGAGACAAAAACAAGCATGCTTCCCATATTCTACATAGGTATTCTAAACATCCAGTTTTGACACAGTGTATCACTAAAGGATTCAAGATTTACACAGAAGTAACCGAGCAGGTCACTAAAGCTCTAAACAGACGTGCTCTAATAGATGAGATAATAAACAATGTATCAACTGATGACAATCTCCTATCAAAACTTCCATTAGAAATTAGGGATCTAATTGTTTCACAAGCTGTCATATAG
上游引物MPV-F:5'-TAGAAAATGGAGCAAATGTCA-3';
下游引物MPV-R:5'-CTACGGGCGATGACAGAT-3';
探针MPV-P:5'-ACGCAGTAATGAGTAA-3';
天花病毒株VAR-IND B19R(NC001611):165773-167497
ATGAGTCGTCGTCTGATTTATGTTTTAAATATCAACCGCAAATCAACTCATAAAATACAAGAGAATGAAATATATACATATTTTAGTTATTGCAATATAGACCATACTTCTACAGAACTTGATTTTGTAGTTAAAAACTATGATCTAAACAGACGACAGCCTGTAACTGGGTATACCGCACTACACTGCTATTTGTATAATAATTACTTTACAAACGATGTACTGAAGGTATTATTAAATCATGGAGTGGATGTAACGATAAAACCCAGTAGCGGACATATGCCTATTTATATATTGCTTACTAGATGTTGCAATATTTCACACAATGTAGTGATAGATATGATAAACAAAGATAAAACCCACTTATCGCATAAAGACTATTCCAACCTACTACTAGAGTATATAAAATCTCGTTACATGCTATTGAAGGAAGAGGATATCGATGAGAACATAGTATCAACTTTATTAGATAAGGGAATCGATCCTAACTTTAAACAAGACGGATATACAGCATTACATTATTATTATTTGTGTCTTGCACACGTTTATAAACCAGGCGAGTGTAGAAAACCGATAACGATAAAAAAGGCCAAGCGAATTATTTCTTTGTTTATACAACATGGAGCTAATCTAAACGCGTTAGATAATTGTGGTAATACACCATTCCATTTGTATCTTAGTATTGAAATGTGTAATAATATCCATATGACTAAAATGCTGTTGACTTTTAATCCGAATTTCGAAATATGTAATAATCATGGATTAACGCCTATACTATGTTATATAACTTCCGACTACATACAACACGATATTCTTGTCATGTTGATACATCACTATGAAACAAATGTTGGAGAAATGCCTATAGATGAGCGTCGTATGATCGTATTCGAGTTTATCAAAACATATTCTACACGTCCGTTAGATTCGATAACTTATTTGATGAATAGGTTTAAAAATATAGATATTCATACCCGCTATGAAGGAAAGACATTATTACACATAGCATGTGAATATAATAATACACACGTAATAGATTATCTTATACGTATTAACGGAGATATAAATGCGTTAACCGACAATAACAAACACGCTATCCAACTCATTATAGATAACAAAGAAAATTCCCAGTATACCATCGATTGTTTATTGTATATACTTAGATATATTGTAGATAAGAATGTGATAAGATCGTTGGTGGATCAACTTCCATATCTACCTATCTTCGATATAAAATCATTTGAGAAATTCATATCATACTGTATACTTTTAGATGACACATTTTACGATAGACACGTTCAGAATCGCGATTCTAAAACTTATCGATACACGTTTTCAAAATACATATCATTCGATAAATACGATAGTATAATAACTAAATGCTATGAAGAAACAATATTGCTCAAACTATCCACTGTTCTAGACACAACACTATATTCTGTTTTAAGATGTCATAATTCGAGAAAGTTAAAAAGATACCTCAGCGTGTTAAAAAAATATAATAATGATAAGTCCTTCAAAATATATTCTAATATTATGAATGAGAGATACCTTAATGTCTATTATAAAGATATGTATGTGTCAAAAGTATACGATAAACTATTTCCTGTTTTCACAGATAAAAAATGTCTACTAACATTACTACCTTCAGAAATTATATATGAAATATTATACATGTTAACAATTTACGATCTTTATAATATATCGTATCCACCTACCAAAGTATAG
上游引物VAR-F:5'-AATATTATGAATGAGAGATACCTTAATGT-3';
下游引物VAR-R:5'-ACATTTTTTATCTGTGAAAACAGG-3';
探针序列VAR-P:5'-TGTGTCAAAAGTATACGA-3';
水痘-带状疱疹病毒株VZV ORF63(NC_001348 ):complement(118480..119316)
atgttttgca cctcaccggc tacgcggggc gactcgtccgagtcaaaacccggggcatcggttgatgtta acggaaagat ggaatatgga tctgcaccaggacccctgaacggccgggatacgtcgcggg gccccggcgc gttttgtact ccgggttgggagatccacccggccaggctcgttgaggaca tcaaccgtgt ttttttatgt attgcacagt cgtcgggacgcgtcacgcgagattcacgaa gattgcggcgcatatgcctc gacttttatc taatgggtcgcaccagacagcgtcccacgt tagcgtgctgggaggaattg ttacagcttc aacccacccagacgcagtgcttacgcgcta ctttaatgga agtgtcccatcgaccccctc ggggggaagacgggttcattgaggcgccga atgttccttt gcataggagc gcactggaatgtgacgtatctgatgatggtggtgaagacg atagcgacga tgatgggtct acgccatcggatgtaattgaatttcgggattccgacgcgg aatcatcgga cggggaagac tttatagtggaagaagaatcagaggagagcaccgattctt gtgaaccaga cggggtaccc ggcgattgttatcgagacggggatgggtgcaacaccccgt ccccaaagag accccagcgt gccatcgagcgatacgcgggtgcagaaaccgcggaatata cagccgcgaa agcgctcacc gcgttgggcgaggggggtgtagattggaagcgacgtcgac acgaagcccc gcgccggcat gatataccgc ccccccatggcgtgtag
上游引物VZV-F:5'-GAGATTCACGAAGATTGCGG-3';
下游引物VZV-R:5'-CCTCCCAGCACGCTAACG-3';
探针VZV-P:5'-CGACTTTTATCTAATGGG-3';
单纯疱疹病毒I型病毒株HSV 1 virionglycoprotein K糖蛋白UL53(X14112):112179..113195
at gctcgccgtc cgttccctgcagcacctctc aaccgtcgtcttgataacgg cgtacggcctcgtgctcgtg tggtacaccgtcttcggtgc cagtccgctg caccgatgtatttacgcggt acgccccaccggcaccaacaacgacaccgc cctcgtgtgg atgaaaatga accagaccctattgtttctgggggccccgacgcacccccc caacgggggc tggcgcaacc acgcccatatctgctacgccaatcttatcgcgggtagggt cgtgcccttc caggtcccac ctgacgccatgaatcgtcggatcatgaacgtccacgaggc agttaactgt ctggagaccc tatggtacac acgggtgcgtctggtggtcgtagggtggtt cctgtatctggcgttcgtcg ccctccacca acgccgatgtatgtttggcgtcgtgagtcc cgcccacaag atggtggccc cggccacctacctcttgaactacgcaggccgcatcgtatc gagcgtgttc ctgcagtacc cctacacgaaaattacccgcctgctctgcgagctgtcggt ccagcggcaa aacctggttc agttgtttgagacggacccggtcaccttcttgtaccaccg ccccgccatc ggggtcatcg taggctgcgagttgatgctacgctttgtggccgtgggtct catcgtcggc accgctttca tatcccggggggcatgtgcgatcacataccccctgtttct gaccatcacc acctggtgtt ttgtctccaccatcggcctgacagagctgtattgtattct gcggcggggc ccggccccca agaacgcagacaaggccgccgccccggggcgatccaaggg gctgtcgggc gtctgcgggc gctgctgttccatcatcctctcgggcatcgcagtgcgatt gtgttatatc gccgtggtgg ccggggtggtgctcgtggcgcttcactacgagcaggagat ccagaggcgc ctgtttgatg tatga
上游引物HSV1-F:5'-GTCTGGTGGTCGTAGGGTG-3';
下游引物HSV1-R:5'-ATCTTGTGGGCGGGACT-3';
探针HSV1-P:5'-TGTATCTGGCGTTCG-3';
人源内参基因GAPDH基因
>NG_007073.2:7681-7980Homo sapiens glyceraldehyde-3-phosphatedehydrogenase (GAPDH), RefSeqGene onchromosome 12
TGGGTGTGAACCATGAGAAGTATGACAACAGCCTCAAGATCATCAGGTGAGGAAGGCAGGGCCCGTGGAGAAGCGGCCAGCCTGGCACCCTATGGACACGCTCCCCTGACTTGCGCCCCGCTCCCTCTTTCTTTGCAGCAATGCCTCCTGCACCACCAACTGCTTAGCACCCCTGGCCAAGGTCATCCATGACAACTTTGGTATCGTGGAAGGACTCATGGTATGAGAGCTGGGGAATGGGACTGAGGCTCCCACCTTTCTCATCCAAGACTGGCTCCTCCCTGCCGGGGCTGCGTGCAA
上游引物Human IC-F:5'-ATGGACACGCTCCCCTGAC-3';
下游引物Human IC-R:5'-TGAGTCCTTCCACGATACCAAA-3';
探针Human IC-P:5'-GCAATGCCTCCTGCACCACCAA-3'。
实施例2
1.标本采集
(1)疱疹表面或渗出液拭子:使用无菌聚酯或涤纶拭子用力涂抹病变部位,以确保收集足够的病毒DNA,将拭子放置无菌收集管中。
(2)疱疹液:用酒精棉球对皮损处进行消毒,并自然干燥。用无菌针头和注射器吸取液体,放置于1.5-2.0mL的无菌O型橡胶圈的螺旋盖塑料管。
(3)咽拭子:用2根聚丙烯纤维头的塑料杆拭子同时擦拭双侧咽扁桃体及咽后壁,将拭子放入无菌采样液管中,尾部弃去,旋紧管盖。
(4)血液标本:采集量5mL,一空腹为佳,使用含有EDTA抗凝剂的真空采血管采集血液标本5mL后,室温静置30分钟,1500-2000rpm离心10分钟,分别收集血浆和血液中细胞于无菌螺旋口塑料管中。
(5)血清标本:用真空负压采血管采集血液标本5mL,室温静置30分钟,1500-2000rpm离心10分钟,收集血清于无菌螺旋口塑料管中。
2.标本保存
标本在收集后1小试内冷藏(2-8℃)或冷冻(-20℃或更低),并尽快进行检测。能在一周以内检测的标本可置于4℃保存;无法检测的标本,短期可在-20℃中保存,长期置于-70℃以下保存(收集后大于60天)。避免反复冻融。
3.标本处理
(1)临床标本
(2)疱疹液:将疱疹液用1mL的PBS重悬取200μL标本;
(3)咽拭子、其他拭子:如拭子在样本保存液中可直接取200uL样本,若为干拭子可用1mL的PBS重悬后取200μL标本;
(4)全血或血清:直接取200μL标本;
(5)上述标本、阴性对照、假病毒阳性对照各取200μL加入核酸提取试剂在全自动核酸提取仪上提取病原体核酸。
4.反应体系配制
(1)每管PCR反应组成
(2)加入模板
本试剂检测模式为每份标本单管检测,配制完成的反应液20μL/管分装至八联管中,加入10μL核酸模板;阴性对照、阳性对照提取物参考标本操作处理和加样。
阴性对照:生理盐水。
阳性对照:含有猴痘病毒、天花病毒、单纯疱疹病毒I型、水痘-带状疱疹病毒靶序列的假病毒溶液。
3.荧光PCR扩增
(1)反应体系(30μL):PCR扩增反应液20μL+核酸模板10μL。
(2)反应程序:50℃ 2min;95℃ 3min;95℃ 10sec、60℃ 30sec,40个循环(60℃时采集荧光信号)。
4.结果判断
(1)质控标准:质控检测结果必须符合下述条件:
阴性对照:
FAM、VIC、ROX、CY5和CY5.5荧光通道下均无Ct值,或Ct值=40;
阳性对照:
FAM、VIC、ROX、CY5荧光通道下,有明显的S型扩增曲线,且Ct值均≤32;CY5.5荧光通道有扩增曲线;
以上两个条件必须在一次实验中同时满足,否则,本次实验无效,全部实验应重新进行。
(2)阴、阳性结果判定
实验有效的前提下,阴、阳性结果判断如下表:
实施例3检测不同类型样本
本实施例使用假病毒模拟不同类型的临床阳性样本,包括血清、血液标本、咽拭子、疱疹液、疱疹表面或渗出液拭子,按照各自样本处理方法提取核酸,使用上海宏石医疗科技有限公司Slan-96型号PCR仪进行效果检测,结果如下:
上述检测结果表明,本发明方法对模拟的血清、血液标本、咽拭子、疱疹液、疱疹表面拭子、渗出液拭子均有很好的检测效果。
实施例4特异性检测
使用上海宏石医疗科技有限公司Slan-96型号PCR仪,检测10份健康志愿者咽拭子标本、其他相关病原体阳性标本或培养物、灭活病毒疫苗、假病毒阳性对照和阴性对照,检测结果如下:
上述检测结果表明,健康人群咽拭子样本和其他病原体均未检出非特异性扩增,本发明的引物探针组合特异性良好。
实施例5灵敏度检测
1.试剂检出限的确定
将假病毒阳性对照稀释成500Copies/mL和200Copies/mL两个浓度,作为灵敏度确定的模板,按照实施例2中样本处理方法处理假病毒,设置反应程序进行扩增检测,各重复10个反应,选择能稳定检出的浓度作为本试剂的最低检出浓度。反应结果如下:
在10次重复实验中,假病毒浓度为500Copies/mL 时全部能检出,表明500Copies/mL浓度为稳定检出浓度,本试剂盒的灵敏度为500Copies/mL。
2.试剂灵敏度的确认
将假病毒阳性对照稀释成500Copies/mL作为模板,使用本试剂按照说明书内容进行样本处理和检测,重复20个反应,确认本试剂的灵敏度。结果如下:
实施例6精密性检测
使用三批试剂,以浓度5000copies/mL假病毒为模板,重复检测10次,对试剂批内精密性进行评估。
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上述三批试剂精密性实验,结果表明本试剂均具有很高的精密性,每批试剂批内变异系数小于5%。
实施例7抗干扰性检测
本试剂用于检测可引起发热出疹性疾病病毒的核酸,临床使用的样本为血清、血液标本、咽拭子、疱疹液、疱疹表面或渗出液拭子,而样本中潜在的一些物质可能会对检测结果产生影响,需要对其进行评估,有关干扰物质主要包括:血红蛋白、粘液及常用药物(左氧氟沙星、阿奇霉素、扑热息痛、对乙酰氨基酚、阿司匹林)等。
本试验使用假病毒模拟临床阳性样本,在假病毒中加入干扰物质,设置不加干扰物质的假病毒作为对照同时检测分析,使用核酸提取试剂提取样本核酸,评价干扰物质对产品检测效果的影响,具体结果如下表:
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通过上述样本模拟实验,样本中含有的干扰物质血红蛋白、粘蛋白,及常用药物(左氧氟沙星、阿奇霉素、扑热息痛、对乙酰氨基酚、阿司匹林)在样本的核酸提取过程中即可被去除,结果数据表明试剂检测具有很好的重复性和稳定性,即临床样本中带有的干扰物质血红蛋白、粘蛋白,及常用药物(左氧氟沙星、阿奇霉素、扑热息痛、对乙酰氨基酚、阿司匹林)对本试剂检测效果无干扰影响。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (1)
1.非疾病诊断目的的检测引起发热出疹性疾病病毒的方法,其特征在于,所述方法包括使用组合物进行荧光PCR扩增的步骤,所述组合物包括用于检测猴痘病毒、天花病毒、单纯疱疹病毒I型和水痘-带状疱疹病毒的引物和探针,其中,
检测猴痘病毒的引物和探针为:
SEQ ID NO.1:5'-TAGAAAATGGAGCAAATGTCA-3';
SEQ ID NO.2:5'-CTACGGGCGATGACAGAT-3';
SEQ ID NO.3:5'-ACGCAGTAATGAGTAA-3';
检测天花病毒的引物和探针为:
SEQ ID NO.4:5'-AATATTATGAATGAGAGATACCTTAATGT-3';
SEQ ID NO.5:5'-ACATTTTTTATCTGTGAAAACAGG-3';
SEQ ID NO.6:5'-TGTGTCAAAAGTATACGA-3';
检测水痘-带状疱疹病毒株的引物和探针为:
SEQ ID NO.7:5'-GAGATTCACGAAGATTGCGG-3';
SEQ ID NO.8:5'-CCTCCCAGCACGCTAACG-3';
SEQ ID NO.9:5'-CGACTTTTATCTAATGGG-3';
检测单纯疱疹病毒I型的引物和探针为:
SEQ ID NO.10:5'-GTCTGGTGGTCGTAGGGTG-3';
SEQ ID NO.11:5'-ATCTTGTGGGCGGGACT-3';
SEQ ID NO.12:5'-TGTATCTGGCGTTCG-3';
所述组合物还包括人源内参基因GAPDH的引物和探针:
SEQ ID NO.13:5'-ATGGACACGCTCCCCTGAC-3';
SEQ ID NO.14:5'-TGAGTCCTTCCACGATACCAAA-3';
SEQ ID NO.15:5'-GCAATGCCTCCTGCACCACCAA-3';
所述探针的5’端标记荧光基团,3’端标记淬灭基团;
所述荧光基团包括FAM、VIC、ROX、CY5和CY5.5;
检测猴痘病毒、天花病毒、单纯疱疹病毒I型和水痘-带状疱疹病毒的探针所标记的荧光基团彼此互不相同且互不干扰;
所述组合物还包括阳性对照、阴性对照,10×PCR Buffer、 MgCl2、 dUTP/dNTPs Mix、BSA、Taq DNA聚合酶、UDG酶和灭菌超纯水;
所述PCR扩增步骤为:50℃ 2min;95℃ 3min;95℃ 10sec、60℃ 30sec,40个循环。
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