CN116287434B - 一种呼吸道症候群病原体核酸检测试剂盒 - Google Patents
一种呼吸道症候群病原体核酸检测试剂盒 Download PDFInfo
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Abstract
本发明属于生物检测领域,具体涉及吸道症候群病原体核酸检测试剂盒。本发明的检测试剂盒可以检测多达39种病原体微生物,包括7种人冠状病毒,甲型流感病毒3种亚型,乙型流感病毒2种亚型,副流感病毒3种亚型,呼吸道合胞疱疹病毒2种亚型,鼻病毒,人偏肺病毒,丙型流感病毒,肠道病毒2种,人巨细胞病毒,腺病毒,EB病毒,肺炎支原体,肺炎衣原体,沙眼衣原体,鹦鹉热衣原体,Q热立克次体,肺炎链球菌,肺炎克雷伯菌,金黄色葡萄球菌,铜绿假单胞菌,A族链球菌,耐甲氧西林葡萄球菌,鲍曼不动杆菌,嗜肺军团菌,流感嗜血杆菌。本发明具有检测病原体数量多,病原体类型广,特异性好、便于运输和保存的特点。
Description
技术领域
本发明属于生物检测领域,具体涉及吸道症候群病原体核酸检测试剂盒。
背景技术
急性呼吸道感染是临床最常见的疾病之一,发病率居急性传染病的首位,—般由细菌和病毒引起,其中大约90%为呼吸道病毒所导致,这些病原体感染都会导致发热、咳嗽、肺炎或白细胞不明显升高等类似临床症状。在人群中,尤其是儿童中,急性呼吸道感染往往是病毒和细菌混合感染或者继发感染,或单一及多种病毒感染,随着病情的持续发展,混合感染现象较为普遍。这给患者病情的控制和病原体的确定增加了难度。
呼吸道感染引起的临床症状和体征都较为相似,其临床表现主要为鼻炎、咽炎、喉炎、扁桃体炎等症状,严重的可引起气管炎、支气管炎及肺炎等,但不同病原体引起的感染,其治疗方法、疗效和病程也不尽相同。目前已证明,大部分呼吸道疾病是由细菌外的病原体引起,其中以呼吸道病毒最常见。呼吸道感染性疾病具有相似的临床症状和流行特征,严重的可能导致重症肺炎、呼吸衰竭,甚至死亡,因此,早诊断、早治疗、早隔离是防止的根本。而只有明确鉴别引起呼吸道感染的病原体后,才便于进行针对性的治疗,提高疗效、降低患者负担,并有效减少抗生物的滥用。
目前常用的检测呼吸道病原体的检测方法:培养法、血清学检测、分子生物学检测方法等。培养法是诊断细菌性感染和病毒感染最可靠的方法,也是诊断的金标准,但这种方法复杂、操作繁琐、周期长、灵敏度低,对样本的要求比较高,而且易出现假阴性的结果。血清学检测是基于抗原抗体反应原理进行检测,检测周期短,但特异性和灵敏度低,易出现假阴性和假阳性的结果,具有一定的局限性。荧光PCR法是目前临床应用较多的病原早期快速诊断的分子生物学方法,但一次通常只能检测一种病原。基因芯片技术可以实现多病原同时检测,但现有芯片技术基本都是扩增后再杂交,操作较为复杂,耗时较长,而且有个开管取PCR产物的过程,这样使高浓度产物暴露空气中,很容易造成样本间的交叉污染。
由此可见,通过临床症状和常规的实验室检测很难鉴别确定所感染的病毒种类,而且病毒的培养条件较苛刻,造成培养阳性率低,甚至有些病毒在目前条件下尚不能培养,这给呼吸道感染患者及临床医生带来很大困扰。
因此,本领域迫切需要一种简便、快速、客观的检测多种呼吸道病原体的方法,同时满足医院门口患者和住院患者检测需求,从而实现明确病原、及早诊断、合理指导临床用药。
发明内容
本发明提供一种呼吸道症候群病原体核酸检测试剂盒,使用5色荧光技术,可以检测多达39种病原体微生物,具体包括人冠状病毒(HCoV-SARS、HCoV-NL63、HCoV-HKU1、HCoV-229E、HCoV-OC43、MERS-CoV、SARS-CoV-2)7种,甲型流感病毒3种亚型(H1N1、H5N1、H7N9),乙型流感病毒2种亚型(Victoria型和Yamagata 型),副流感病毒3种亚型(1、2、3型),呼吸道合胞疱疹病毒2种亚型(A型和B型),鼻病毒,人偏肺病毒,丙型流感病毒,肠道病毒2种(EV71和CA16),人巨细胞病毒,腺病毒,EB病毒,肺炎支原体,肺炎衣原体,沙眼衣原体,鹦鹉热衣原体,Q热立克次体,肺炎链球菌,肺炎克雷伯菌,金黄色葡萄球菌,铜绿假单胞菌,A族链球菌,耐甲氧西林葡萄球菌,鲍曼不动杆菌,嗜肺军团菌,流感嗜血杆菌。
对于上述多种病原体,本发明针对性的选择靶基因,并设计特异性引物和探针,具体如下:
1.新型冠状病毒(SARS-Cov-2)ORF1ab基因
>NC_045512.2 Severe acute respiratory syndrome coronavirus 2 isolateWuhan-Hu-1, complete genome
gaatgtcttg acactcgttt ataaagttta ttatggtaat gctttagatc aagccatttccatgtgggct cttataatct ctgttacttc taactactca ggtgtagtta caactgtcat
gtttttggcc agaggtattg tttttatgtg tgttgagtat tgccctattt tcttcataac
tggtaataca cttcagtgta taatgctagt ttattgtttc ttaggctatt tttgtacttg
ORF1ab-Fwd:5'-TTCCATGTGGGCTCTTATAATC-3',
ORF1ab-Rwd:5'-TCAACACACATAAAAACAATACCTC-3';
ORF1ab-P:5'FAM-TGTTACTTCTAACTACTCAGGTG-BHQ1 3';
2.冠状病毒HCoV-229E ORF1ab基因
>NC_002645.1 Human coronavirus 229E, complete genome
TGTGGAACCTTGTATTTAACATACTTTCCATGTTTTCATCTTCATTCTCTGTTGCTGCAATGTCAGGTCAAATTTTACTTAATTGTGCATTAGGTGCTTTTGCTATTTTTTGTTGTTTTCTTGTGACAAAGTTTAGACGCATGTTTGGTGACCTTTCTGTAGGTGTTTGCACTGTTGTTGTGGCTGTTTTGCTTAAC
上游引物F:5'-GTTTTCATCTTCATTCTCTGTTGC-3';
下游引物R:5'-TGTCACAAGAAAACAACAAAAAAT-3';
探针P:5'-FAM-CTTAATTGTGCATTAGGTGC-MGB-3';
3.冠状病毒SARS-CoV ORF1ab基因
>AY278488.2 SARS coronavirus BJ01, complete genome
GAGTAAAATTTTGGCTTATGTCAAACCATTCTTAGGACAAGCAGCAATTACAACATCAAATTGCGCTAAGAGATTAGCACAACGTGTGTTTAACAATTATATGCCTTATGTGTTTACATTATTGTTCCAATTGTGTACTTTTACTAAAAGTACCAATTCTAGAATTAGAGCTTCACTACCTACAACTATTGCTAAAAATAGTGTTAAGAG
上游引物F:5'-GAGTAAAATTTTGGCTTATGTCA-3';
下游引物R:5'-CAATAGTTGTAGGTAGTGAAGCTCT-3';
探针P:5'-FAM-ATTCTTAGGACAAGCAGC-MGB-3';
4.冠状病毒HCoV-OC43 ORF1ab基因
>KF530060.1 Human coronavirus OC43 strain OC43/human/USA/851-15/1985,complete genome-ORF1ab基因
AGTTCCTTGTGCTGGAAGGCGTGTTACATTTAAGGAACAGCCTACAGTAAAGGAGATTATAAGCATGCCTAAGATTATTAAGGTTTTTTATGAGCTCGACAACGATTTTAATACTATTTTAAATACTGCATGTGGAGTGTTTGAAGTGGATGATACTGTGGATATGGAGGAATTTTATGCTGTGGTGATTGATGCCATAGAAGAGAAACT
上游引物F:5'-CTTGTGCTGGAAGGCGT-3';
下游引物R:5'-TTCAAACACTCCACATGCAG-3';
探针P:5'-FAM-AAGGAACAGCCTACAGTAA-MGB-3';
5.冠状病毒MERS-CoV-ORF1ab基因
>KT029139.1 Middle East respiratory syndrome coronavirus isolateMERS-CoV/KOR/KNIH/002_05_2015, complete genome
CTGTCCAAAAAGAGTCAGATGAGTATATTCTGGCTAAAGGGCCGTTACAAGTAGGAGATTCAGTTCTCTTGCAAGGCCATTCTCTAGCTAAGAATATCCTGCATGTCGTAGGCCCAGATGCCCGCGCTAAACAGGATGTTTCTCTCCTTAGTAAGTGCTATAAGGCTATGAATGCATATCCTCTTGTAGTCACTCCTCTTGTTTCAACAGGCA
上游引物F:5'-GTCCAAAAAGAGTCAGATGAGTAT-3';
下游引物R:5'-ACGACATGCAGGATATTCTTAG-3';
探针P:5'-FAM-TTCTGGCTAAAGGGC-MGB-3';
6.冠状病毒HCoV-HKU1-ORF1ab基因
>NC_006577.2 Human coronavirus HKU1, complete genome
GTTTAAGGATTTAGCTATTGAAAATATGTGGTTATCTTATAAGGTGGGTTATAATCAAAGTTTTGTTGATTATTTACTGACCACTATTCCTAAAGCTATTGTTTTGCCTCAAGGTGGTTTTGTAGCTGATTTTGCTTATTGGTTTTTAAACCAGTTTGATATTAATGCGTATGCTAATTGGTGTTGTTTAAAATGTGGTT
上游引物F:5'-GTGGTTATCTTATAAGGTGGGTTA-3';
下游引物R:5'-TACGCATTAATATCAAACTGGTT-3';
探针P:5'-FAM-CCACTATTCCTAAAGC-MGB-3';
7.冠状病毒HCoV-NL63-ORF1ab基因
>JQ765563.1 Human coronavirus NL63 strain NL63/DEN/2009/9, complete
TTTTAGAGATGAATTGGGTGTTCGTGTTTTAGATCAATCTGATAATAATTGTTGGATTAGTACCACACTTATACAGTTGCAACTTACAAAGCTTTTGGATGATTCTATTGAGATGCAATTGTTTAAAGTTGGTAAAGTTGATTCAATTGTTCAAAAGTGTTATGAGTTGTCTCATTTAATTAGTGGTTCACTTGGTGATAGTGGTAAACT
上游引物F:5'-TTGGGTGTTCGTGTTTTAGAT-3';
下游引物R:5'-CTCAATAGAATCATCCAAAAGC-3';
探针P:5'-FAM-TCTGATAATAATTGTTGGATT-MGB-3';
8.甲型H1N1流感病毒-matrix(M1)
TCAGGCCCCCTCAAAGCCGAGATCGCACAGAGACTGGAAAGTGTCTTTGCAGGAAAGAACACAGATCTTGAGGCTCTCATGGAATGGCTAAAGACAAGACCAATCTTGTCACCTCTGACTAAGGGAATTTTAGGATTTGTGTTCACGCTCACCGTGCCCAGTGAGCGAGGACTGCAGCGTAGACGCT
上游引物F:5'-GCCCCCTCAAAGCCGA-3';
下游引物R:5'-CGCTCACTGGGCACGG-3';
探针P:5'-FAM-TCTCATGGAATGGCTAAAGACAA-BHQ1-3';
9.甲型H5N1流感病毒-matrix(M1)基因
上游引物F:5'-GCCCCCTCAAAGCCGA-3';
下游引物R:5'-CGCTCACTGGGCACGG-3';
探针P:5'-HEX-TCTCATGGAGTGGCTAAAGACAA-BHQ1-3';
10.甲型H7N9流感病毒 matrix(M1)基因
上游引物F:5'-GCCCCCTCAAAGCCGA-3';
下游引物R:5'-CGCTCACTGGGCACGG-3';
探针P:5'-ROX-CATGGAGTGGATAAAGACAAGACC-BHQ2-3';
11.乙型流感病毒Victoria HA基因B/Brisbane/60/2008
TTTTGCAAATCTCAAAGGAACAGAAACCAGGGGGAAACTATGCCCAAAATGCCTCAACTGCACAGATCTGGACGTAGCCTTGGGCAGACCAAAATGCACGGGGAAAATACCCTCGGCAAGAGTTTCAATACTCCATGAAGTCAGACCTGTTACATCTGGGTGCTTTCCTATAATGCACGACAGAACAAAAAT
上游引物F:5'-TTGCAAATCTCAAAGGAACA-3';
下游引物R:5'-TGCATTATAGGAAAGCACCC-3';
探针P:5' FAM-CTTGCCGAGGGTATTTTCC-BHQ1-3';
12乙型流感病毒Yamagata-HA基因 B/Wisconsin/01/2010
TTTTGCAAATCTCAAAGGAACAAGGACCAGAGGGAAACTATGCCCGGACTGTCTCAACTGTACAGATCTGGATGTGGCCTTGGGCAGGCCAATGTGTGTGGGGACCACACCTTCTGCTAAAGCTTCAATACTCCACGAGGTCAGACCTGTTACATCCGGGTGCTTTCCTATAATGCACGACAGAACAAAAAT
上游引物F:5'-TTGCAAATCTCAAAGGAACA-3';
下游引物R:5'-TGCATTATAGGAAAGCACCC-3';
探针P:5'-FAM-TTAGCAGAAGGTGTGGTCCC-BHQ1-3';
13.丙型流感病毒Influenza C M1基因
CAGACGACTACACACCAGACATCCGAATAGGAACAATCACAGCTTGGTTGAGATGCAAAAACAAGAAAAGTGAAAGATACAGGAGTAATGTCTCAGAAAGTGGAAGAACAGCTTTAAAAATTCATGAAGTAAGAAAAGCCAGCACA
上游引物F:5'-GACGACTACACACCAGACATCC-3';
下游引物R:5'-AATTTTTAAAGCTGTTCTTCCAC-3';
探针P:5'-FAM-CAAGAAAAGTGAAAGATACAGGAG-BHQ1-3';
14.副流感病毒1型-HN基因
JF416792.1 Human parainfluenza virus 1 strain KPIV-07-2hemagglutinin-neuraminidase (HN) gene, complete cds
AGTGCCAAAACAATCAAAGAGACAATCACAGAATTAATCAGACAAGAAGTGATATCAAGGACTATAAACATACAAAGTTCAGTACAAAGCGGGATCCCAATATTGTTAAACAAGCAAAGCAGAGATCTCACACAATTAATAGAGAAGTCATGCAACAAACAGGAATTGGCTCAGATATGCGAG
上游引物F:5'-ACAATCAAAGAGACAATCACAGAA-3';
下游引物R:5'-GATCTCTGCTTTGCTTGTTTAAC-3';
探针P:5'-FAM-AGAAGTGATATCAAGGAC-MGB-3';
15.副流感病毒2型-HN基因
JF416794.1 Human parainfluenza virus 2 strain KPIV-06-26hemagglutinin-neuraminidase (HN) gene, complete cds
TTCAGGACTATGAAAACCATTTACCTAAGTGATGGAATCAATCGCAAAAGCTGTTCAGTCACTGCTATACCAGGAGGTTGTGTCTTGTACTGTTATGTAGCTACAAGATCTGAGAAAGAAGATTATGCCACAACTGACCTAGCTGAACTGAGACTTGCTTTCTATTATTATAATGAT
上游引物F:5'-CTAAGTGATGGAATCAATCGC-3';
下游引物R:5'-AGGTCAGTTGTGGCATAATCTT-3';
探针P:5'-FAM-TGCTATACCAGGAGGTTGTGTC-MGB-3';
16.副流感病毒3型-HN基因
Z26523.1 Human parainfluenza virus type 3 HN gene for hemagglutinin-neuraminidase
AAGTCATGTTCTCTAGCACTCCTAAATACAGATGTATATCAACTGTGTTCAACTCCCAAAGTTGATGAAAGATCAGATTATGCATCATCAGGCATAGAAGATATTGTACTTGATATTGTCAATTATGATGGCTCAATCTCAACAACAAGATTTAAGAATAATAA
上游引物F:5'-AAGTCATGTTCTCTAGCACTCCT-3';
下游引物R:5'-TTGTTGAGATTGAGCCATCATA-3';
探针P:5'-FAM-TCAAGTACAATATCTTCTA-MGB-3';
17.呼吸道合胞疱疹病毒A 型-F基因
KY296797.1 Human respiratory syncytial virus A strain SH10-17 fusionglycoprotein (F) gene, complete cds
GTCAGTGTCTTAACCAGCAAAGTGTTAGACCTCAAAAACTATATAGATAAACAGTTGTTACCTATTGTTAACAAGCAAAGCTGCAGCATATCAAACATTGAAACTGTGATAGAGTTCCAACAAAAGAACAACAGACTACTAGAGATTACCAGAGAATTTAGTGTTAATGCAGGTGTAACTACACCTGTAAGCACTT
上游引物F:5'-GTYTTAACCAGCAAAGTGTTAGA-3';
下游引物R:5'-CACCTGCATTRACACTAAATTCT-3';
探针P:5'-FAM-AGATAAACAGTTGTTACCTA-MGB-3';
18.呼吸道合胞疱疹病毒B型-F基因
KY296706.1 Human respiratory syncytial virus B strain Hunan13-04fusion glycoprotein (F) gene, complete cds
GTCAGTGTTTTAACCAGCAAAGTGTTAGATCTCAAGAATTATATAAACAACCAATTATTACCTATAGTAAATCAACAGAGTTGTCGCATATCCAACATTGAAACAGTTATAGAATTCCAGCAGAAGAACAGCAGATTGTTGGAAATCACCAGAGAATTTAGTGTCAATGCAGGTGTAACGACACCTTTAAGCACTT
上游引物F:5'-GTYTTAACCAGCAAAGTGTTAGA-3';
下游引物R:5'-CACCTGCATTRACACTAAATTCT-3';
探针P:5'-TTCCAACAATCTGCTGTT-3';
19.鼻病毒HRV- 5’UTR基因
GQ223229.1 Human rhinovirus A isolate N13, complete genome
ACAAGGTGTGAAGAGCCCCGTGTGCTCACTTTGAGTCCTCCGGCCCCTGAATGTGGCTAACCTTAACCCTGCAGCTAGTGCATACAATCCAGTGTGTGGCTAGTCGTAATGAGCAATTGCGGGATGGGACCAACTACTTTGGGTGTCCGTGTTTC
上游引物F:5'-TTGAGTCCTCCGGCCC-3';
下游引物R:5'-AAACACGGACACCCAAAGTA-3';
探针P:5'-FAM-GGCTAACCYTAACCCT-MGB-3';
20.人偏肺病毒--N基因+P基因
KC562235.1 Human metapneumovirus strain HMPV/USA/C2-202/2004/B,complete genome
TGAGTGGTGACAATCAAGATGATTATGAGTAATTAAAAAACTGGGACAAGTCAAAATGTCATTCCCTGAAGGAAAGGATATCCTGTTCATGGGTAATGAAGCAGCAAAAA
上游引物F:5'-YGACARTCAARATGATTATGAGTAA-3';
下游引物R:5'-GCTGCTTCATTACCCATGAA-3';
探针P:5' FAM-TGGGACAAGTCAAAAT-MGB-3';
21.肠道病毒EV71-VP1基因
GU366191.1| Human enterovirus 71 strain Henan10-08-China, completegenome
TGATGGGCACGTTCTCAGTGCGGACTGTGGGGACCTCCAAGTCCAAGTACCCTTTAGTGGTTAGGATTTACATGAGAATGAAGCACGTCAGGGCGTGGATACCTCGCCCGATGCGTAACCAGAACTACCTATTCAAAGCCAACCCAAATTATGCTGGCAACT
上游引物F:5'-CGTTCTCAGTGCGGACTG-3';
下游引物R:5'-TAGGTAGTTCTGGTTRCGCATY-3';
探针P:5'-FAM-TCCAAGTCCAAGTACCCTTTAG-BHQ1-3';
22.肠道病毒CA16-VP1基因
HE572994.1| Human coxsackievirus A16 partial gene for polyprotein,VP1 protein region, isolate CF279014_FRA10, genomic RNA
GGCGGAAATGCGAGTTGTTTACCTACATGCGCTTTGATGCTGAATTCACATTTGTTGTGGCTAAACCTAATGGTGAGCTAGTCCCCCAATTGCTACAGTACATGTATGTCCC
上游引物F:5'-GGCGGAAATGCGAGTT-3';
下游引物R:5'-GTACTGYAGYAATTGGGGGA-3';
探针P:5'-FAM-ATGCGCTTTGATGC-MGB-3';
23.人巨细胞病毒-IE基因
M21295.1 Human cytomegalovirus (HCMV) major immediate-early protein(IE) gene, complete cds
GCTATGTCTTAGAGGAGACTAGTGTGATGCTGGCCAAGCGGCCTCTGATAACCAAGCCTGAGGTTATCAGTGTAATGAAGCGCCGCATTGAGGAGATCTGCATGAAGGTCTTTGCCCAGTACATTCTGGGGGCCGATCCTCTGAGAGTCTGCTCTCCTAG
上游引物F:5'-AGAGGAGACTAGTGTGATGCTG-3';
下游引物R:5'-AATGTACTGGGCAAAGACCT-3';
探针P:5'-ATAACCAAGCCTGAGG-3';
24.腺病毒--Hexon基因
AB330088.1| Human adenovirus 7 gene for hexon, complete cds GACATGACTTTTGAGGTGGACCCCATGGATGAGCCCACACTTCTCTATGTTCTGTTCGAAGTTTTCGACGTTGTGCGCATCCACCAGCCGCACCGCGGCGTCATCGAGGCCGTCTACCTGCGTACGCCGTTCTCGGCCGGTAACGCCACCAC
上游引物F:5'-GACATGACYTTYGAGGTGGA-3';
下游引物R:5'-GTAGACGGCCTCGATGA-3';
探针P:5'-FAM-CCCATGGATGAGCCCAC-BHQ1-3';
25.EB病毒- LMP1基因
D10059.1 Human herpesvirus 4 gene for LMP1, complete cds
AATCTGACTCTAACTCCAACGAGGGCAGACACCACCTGCTCGTGAGTGGAGCCGGCGACGGACCCCCACTCTGCTCTCAAAACCTAGGCGCACCTGGAGGTGGTCCTGACAATGGCCCACAGGACCCTGACAACACTGATGACAATGGCCCACAGGACCCTGACAACACTGATGACAATGGCCCACA
上游引物F:5'-CTAACTCCAACGAGGGCAG-3';
下游引物R:5'-GGACCACCTCCAGGTGC-3';
探针P:5'-FAM-TTGAGAGCAGAGTGGG-BHQ1-3';
26.肺炎支原体MP-P1基因
EF656612.1 Mycoplasma pneumoniae isolate Mp3896 protein P1 (P1) gene,complete cds
CGCCACACCAATGCCATCAACCCGCGCTTAACCCCGTGAACGTATCGTAACACGAGCTTTTCCTCCCTCCCCCTCACGGGTGAAAATCCCGGGGCGTGGGCCTTAGTGCGCGACAACAGCGCTAAGGGCATCACTGCCGGCAGTGGCAGTCAACAAAC
上游引物F:5'-ACCAATGCCATCAACCC-3';
下游引物R:5'-AGTGATGCCCTTAGCGC-3';
探针P:5'-FAM-TTAACCCCGTGAACG-MGB-3';
27.肺炎衣原体CP-主要外膜蛋白(MOMP)基因
>AF131889.1 Chlamydia pneumoniae major outer membrane protein (MOMP)gene, complete cds
上游引物CP-F: 5'-TTCCCTTGCCAACAGACG-3';
下游引物CP-R:5'-GGCTGAGCAATGCGGATGT-3';
探针CP-P:5'FAM-CGACCATCAATTATC-MGB 3';
28.沙眼衣原体CT-omp1基因
X62918.1 C.trachomatis (D/B-120) omp1 gene for major outer membraneprotein
CAATGCAAATCGTTTCCTTGCAATTGAACAAGATGAAATCTAGAAAATCTTGCGGTATTGCAGTAGGAACAACTATTGTGGATGCAGACAAATACGCAGTTACAGTTGAGACTCGCTTGATCGATGAGAGAGCAGCTCACGTAAATGCACAATTCCGCTTCTAA
上游引物F:5'-AATGCAAATCGTTTCCTTG-3';
下游引物R:5'-TTGTGCATTTACGTGAGCTG-3';
探针P:5'-FAM-AAATCTAGAAAATCTTGCG-MGB-3';
29.鹦鹉热衣原体 MOMP基因
EU856036.1 Chlamydophila psittaci strain SP16 MOMP gene, complete cds
TACGGGTTCCGCTCTCTCCTTACAAGCCTTGCCTGTAGGGAACCCAGCTGAACCAAGTTTATTAATCGATGGCACTATGTGGGAAGGTGCTTCAGGAGATCCTTGCGATCCTTGCGCTACTTGGTGTGACGCCATTAGCATCCGCGCAGGATACTACGGAG
上游引物F:5'-TACGGGTTCCGCTCTCTC-3';
下游引物R:5'-GCGTCACACCAAGTAGMG-3';
探针P:5'-FAM-CAAGCCTTGCCTGTAG-MGB-3';
30. Q热立克次体(Q热柯克斯体、贝纳柯克斯体)转位酶IS1111a基因
M80806.1 Coxiella burnetii transposase (IS1111a) gene, complete cds
GTTGCAAGAATACGGACTCACGATGGCGCGTGGTGCCAAGCGATTTTATGAAGAGCTCCCGTTGATTTTAGCGAGCGAAGCGGTGGGATTAACACCGCGGATGAAACGGGTGTTGAATTGTTTGTATACCGAATTGTTGAACCGGGACGAAGCG
上游引物F:5'-AATACGGACTCACGATGGC-3';
下游引物R:5'-AACACCCGTTTCATCCG-3';
探针P:5'-FAM-CCAAGCGATTTTATGA-BHQ1-3';
31. 肺炎链球菌SA-自溶素lytA基因
M13812.1 S.pneumoniae autolysin (lytA) gene, complete cds
ATTGTTGGGAACGGTTGCATCATGCAGGTAGGACCTGTTGATAATGGTGCCTGGGACGTTGGGGGCGGTTGGAATGCTGAGACCTATGCAGCGGTTGAACTGATTGAAAGCCATTCAAC
上游引物F:5'-ATTGTTGGGAACGGTTGC-3';
下游引物R:5'-TTTCAATCAGTTCAACCGCT-3';
探针P:5'-FAM-TAGGACCTGTTGATAATG-BHQ1-3';
32. 肺炎克雷伯菌KPN 16S rRNA基因
FJ436718.1 Klebsiella pneumoniae strain FIUMS1 16S ribosomal RNAgene, complete sequence GGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGC
上游引物F:5'-GACACGGTCCAGACTCCTAC-3';
下游引物R:5'-AAGTGCTTTACAACCCGAA-3';
探针P:5'-FAM-AGCAGTGGGGAATAT-BHQ1-3';
33. 金黄色葡萄球菌
MG970610.1 Staphylococcus aureus aminoacyl transferase (femB) gene,complete cds
GGATTAGTTGATTATTATTTAAAAGAGTTAGATAAATATTTACAGCAACATCAATGTTTATATGTTAAATTAGATCCGTATTGGTTATATCATCTATATGATAAAGATATCGTGCCATTTGAAGGTCGCGAGAAAAATGAT
上游引物F:5'-GAGTTAGATAAATATTTACAGCAACA-3';
下游引物R:5'-GGCACGATATCTTTATCATATAGA-3';
探针P:5' FAM-AATTAGATCCGTATTGGTTA-MGB-3';
34. 铜绿假单胞菌PA(绿脓杆菌) oprI基因
X58714.1 Pseudomonas aeruginosa oprI gene for lipoprotein I (OprI)
TGACCGCTACCGAAGACGCAGCTGCTCGTGCTCAGGCTCGCGCTGACGAAGCCTATCGCAAGGCTGACGAAGCTCTGGGCGCTGCTCAGAAAGCTCAGCAGACTGCTGACGAGGCTAACGAGCGTGCCCTGCGCATGCTGGAAAAAGCCAGCCGCAAG
上游引物F:5'-GACCGCTACCGAAGACG-3';
下游引物R:5'-CGCTCGTTAGCCTCGTC-3';
探针P:5'-FAM-GCTCGTGCTCAGGCTCG-BHQ1-3';
35. 化脓性链球菌(A组链球菌GAS)链球菌溶血素O基因
L26152.1 Streptococcus pyogenes pyrogenic exotoxin B (speB) gene,complete cds
ACCTACTTATAGCGGAAGAGAATCTAACGTTCAAAAAATGGCGATTTCAGAATTGATGGCTGATGTTGGTATTTCAGTAGACATGGATTATGGTCCATCTAGTGGTTCTGCAGGTAGCTCTCGTGTTCAAAGAGCC
上游引物F:5'-ACCTACTTATAGCGGAAGAGAAT-3';
下游引物R:5'-ACGAGAGCTACCTGCAGAAC-3';
探针P:5'-FAM-ATTTCAGAATTGATGGCT-BHQ1-3';
36. 耐甲氧西林葡萄球菌-mecA基因
KC243783.1 Staphylococcus aureus strain TN/CN/1/12 MecA (mecA) gene,complete cds
AGAAGATGGTATGTGGAAGTTAGATTGGGATCATAGCGTCATTATTCCAGGAATGCAGAAAGACCAAAGCATACATATTGAAAATTTAAAATCAGAACGTGGTAAAATTTTAGACCGAAACAATGTGGAATTGGCCAATACAGGAACAGCATATGA
上游引物F:5'-AGTTAGATTGGGATCATAGCGT-3';
下游引物R:5'-TTCCACATTGTTTCGGTCTAA-3';
探针P:5'-FAM-AATGCAGAAAGACCAAA-MGB-3';
37.鲍曼不动杆菌-OXA51基因
KF048907.1| Acinetobacter baumannii strain SR17 beta-lactamase OXA-51-like protein gene, complete cds
AGCAGAGAAAATTAAAAATTTATTTAACGAAGCACACACTACGGGTGTTTTAGTTATCCAACAAGGCCAAACTCAACAAAGCTATGGTAATGATCTTGCTCGTGCTTCGACCGAGTATGTACCTGCTTCGACCTTCAAAATGCTTAATGCTTTGATCGGCC
上游引物F:5'-ACACACTACGGGTGTTTTAGTT-3';
下游引物R:5'-AGCATTAAGCATTTTGAAGGT-3';
探针P:5'-AACTCAACAAAGCTATG-3';
38. 嗜肺军团菌LP-MIP基因
AF095226.1 Legionella pneumophila strain ATCC 33215 macrophageinfectivity potentiator (mip) gene, complete cds
ACATCATTAGCTACAGACAAGGATAAGTTGTCTTATAGCATTGGTGCCGATTTGGGGAAGAATTTTAAAAATCAAGGCATAGATGTTAATCCGGAAGCAATGGCTAAAGGCATGCAAGACGCTATGAGTGGCGCTCAATTGGCTTTAACCGAA
上游引物F:5'-ACATCATTAGCTACAGACAAGGA-3';
下游引物R:5'-TCGGTTAAAGCCAATTGAG-3';
探针P:5'-FAM-ATAGCATTGGTGCCGATTT-BHQ1-3';
39. 流感嗜血杆菌Hi 荚膜bexA基因
M19995.1 Haemophilus influenzae bexA gene, complete cds
TTGTCGTTTGTATGATGTTGATCCAGACTACGTTACTCGTTTTACAAAAGAGTTTTCTGAATTGGGCGATTATCTTTATGAGCCAGTGAAGAAGTACTCATCAGGGATGAAAGCCCGACTTGCATTTGCTCT
上游引物F:5'-GTCGTTTGTATGATGTTGATCC-3';
下游引物R:5'-CGGGCTTTCATCCCTG-3';
探针P:5'-FAM-ACTCGTTTTACAAAAGA-MGB-3';
人源内参基因GAPDH基因
>NG_007073.2:7681-7980 Homo sapiens glyceraldehyde-3-phosphatedehydrogenase (GAPDH), RefSeqGene on chromosome 12
TGGGTGTGAACCATGAGAAGTATGACAACAGCCTCAAGATCATCAGGTGAGGAAGGCAGGGCCCGTGGAGAAGCGGCCAGCCTGGCACCCTATGGACACGCTCCCCTGACTTGCGCCCCGCTCCCTCTTTCTTTGCAGCAATGCCTCCTGCACCACCAACTGCTTAGCACCCCTGGCCAAGGTCATCCATGACAACTTTGGTATCGTGGAAGGACTCATGGTATGAGAGCTGGGGAATGGGACTGAGGCTCCCACCTTTCTCATCCAAGACTGGCTCCTCCCTGCCGGGGCTGCGTGCAA
上游引物:5'-ATGGACACGCTCCCCTGAC-3';
下游引物:5'-TGAGTCCTTCCACGATACCAAA-3';
探针:5'FAM-GCAATGCCTCCTGCACCACCAA-BHQ1 3'。
本发明中涉及的临床标本类型为鼻/口咽拭子、痰液、肺泡灌洗液。
本发明使用的荧光定量PCR仪包括罗氏LightCycler 480、ABI 7500,上海宏石Slan-96P,西安天隆TL988、96E、48E,Bio-rad CFX96,POCT分子一体机等。
本发明提供一种呼吸道症候群病原体核酸检测试剂,包括8种PCR反应液1-8,扩增酶混合液、阳性对照,阴性对照。
本发明提供的试剂盒可应用于门诊呼吸道疾病患者和住院患者的鼻/口咽拭子、痰液、肺泡灌洗液标本,可用于回顾性验证实验。
本发明技术方案中,所述探针的5’端标记荧光基团,3’端标记淬灭基团,其中,荧光基团可选 FAM、HEX、JOE、TET、CY3、CY5、ROX、Texas等,本发明的优选方案为FAM;淬灭基团可选TAMRA、BHQ(BHQ1、BHQ2、BHQ3)、MGB等,为了检测需要,可将上述探针的5’端的荧光基团,或3’端标记的淬灭基团进行替换。
本发明可以检测39种呼吸道病原体,外加一个人源内参基因,共计40个靶基因,采用5色荧光PCR技术,每管可以检测5个靶基因,检测一人份样本时需要使用整条八联管。下表为40个靶基因的组合设计之一:
上表中仅为靶基因的一种组合方式,还可以将40个靶基因随机分成不同的组,利用本发明的引物、探针均可实现有效的检测。
使用方法:
1.标本处理
(1)临床标本:呼吸道临床标本(咽拭子、液化后的痰液或肺泡灌洗液)直接取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。
(2)阴性对照、假病毒阳性对照: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。
2.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多管检测,即每份标本均需要使用8种PCR反应液。配制完成的8种反应液20μL/管分装至八联管中,每份标本一条八联管。每管分别加入5μL核酸模板,即上述同一份标本提取物;阴性对照、阳性对照提取物参考标本操作处理和加样。
3.荧光PCR扩增
(1)反应体系(25μL):PCR扩增反应液20μL+核酸模板5μL。
(2)反应程序:50℃ 15min;95℃.3min;95℃ 10sec、58℃ 30sec,45个循环(58℃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=45;
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:
本发明的有益效果:
本发明提供一种呼吸道症候群病原体核酸检测试剂盒,使用5色荧光技术,可以检测多达39种病原体微生物,具体包括人冠状病毒(HCoV-SARS、HCoV-NL63、HCoV-HKU1、HCoV-229E、HCoV-OC43、MERS-CoV、SARS-CoV-2)7种,甲型流感病毒3种亚型(H1N1、H5N1、H7N9),乙型流感病毒2种亚型(Victoria型和Yamagata 型),副流感病毒3种亚型(1、2、3型),呼吸道合胞疱疹病毒2种亚型(A型和B型),鼻病毒,人偏肺病毒,丙型流感病毒,肠道病毒2种(EV71和CA16),人巨细胞病毒,腺病毒,EB病毒,肺炎支原体,肺炎衣原体,沙眼衣原体,鹦鹉热衣原体,Q热立克次体,肺炎链球菌,肺炎克雷伯菌,金黄色葡萄球菌,铜绿假单胞菌,A族链球菌,耐甲氧西林葡萄球菌,鲍曼不动杆菌,嗜肺军团菌,流感嗜血杆菌,使用人源基因GAPGH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,具有检测病原体数量多,病原体类型广,特异性好、便于运输和保存的特点。
本发明的试剂盒主要用于上述病原微生物的同时检测或鉴定,为临床提供抗生素用药指导,避免过度用药。目前,基于荧光探针的多重荧光PCR试剂基本都是对同一种类型的核酸进行检测,或都针对DNA类的核酸进行多重荧光PCR,或都针对RNA类的核酸进行多重荧光PCR,且检测的病原体种类较少,未能覆盖呼吸道系统大部分主要病原体。本发明的优点在于检测试剂能同时检测DNA和RNA两种不同类型的核酸,能实现对多种类型核酸的检测,既包括常见呼吸道病原体,也含有非典型肺炎病原体,既能检测病毒,也能检测细菌、支原体、衣原体等病原体,实现一个标本,一次提取,得到多个检测结果,同时也能给反映细菌耐药情况(耐甲氧西林葡萄球菌),扩大应用范围,缩短病原体检测时间,节省试剂和耗材。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1显示实施例1中电泳检测结果;
图2显示实施例2中特异性检测实验的结果;
图3显示实施例3中新型冠状病毒扩增结果;
图4显示实施例3中腺病毒扩增结果;
图5显示实施例3中副流感病毒2型扩增结果;
图6显示实施例3中肺炎链球菌扩增结果;
图7显示实施例3中EB病毒的扩增结果;
图8显示实施例3中呼吸道合胞疱疹病毒A型和肺炎衣原体扩增结果。
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。
一、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)2×PCR反应缓冲液:全称2×Hifair VN MP-Buffer,货号13123A;
(4)扩增酶混合物:全称Hifair VN Enzyme Mix(UDG plus),货号13123B;
(5)DEPC处理水:自制;
(6)核酸提取试剂:型号Ex-DNA/RNA。
(7)10份健康志愿者咽拭子:公司员工。
(8)其他病原体阳性标本或培养物:灭活肺炎支原体体临床标本、灭活肺炎衣原体临床培养物、灭活流感病毒临床标本、灭活呼吸道合胞疱疹病毒临床标本,医院惠赠。
(9)新型冠状病毒核酸检测试剂国家参考品(批号370099-202001),中国食品药品检定研究院。
实施例1引物的设计和筛选
本发明在NCBI数据库中下载上述39种病原体的基因组序列,每种病原体约10条,使用生物学软件BioEdit进行序列对比分析,查找病原体之间没有交叉且特有的保守区域,具体如下:
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在此基础再进一步确定靶序列。
同时,针对具有多种亚型的病原体,根据保守区域序列的具体情况,采用不同的策略设计引物和探针,从而进一步避免各种病原体之间的干扰,如3种甲型流感病毒(H1N、H5N1和H7N9)、2种乙型流感病毒(Victoria和Yamagata)、2种呼吸道合胞病毒(A 型和B型)3种病原体,检测时采用共同引物扩增保守区域,再使用特异探针进行亚型鉴定;而3种副流感病毒(1型、2型和3型)、7种冠状病毒则采用每种亚型特有的引物和探针直接进行亚型鉴定。
引物、探针设计完成后,为了检测引物二聚体和非特异性扩增情况,通过实验再次确认引物的特异性。
(1)普通PCR扩增和电泳检测
本发明配制含有40套引物的反应体系,一孔位不加模板用于检测引物二聚体,其余孔位加入模板。
分子标记物M 显示的8条带从上往下分别为100bp、250bp、500bp、750bp、1000bp、2000bp、3000bp和5000 bp。如图1所示,第1孔没有引物二聚体片段,3-5孔扩增结果仅有目的片段,也没有引物二聚体,表明设计的引物之间均没有形成二聚体,不存在相互干扰而影响扩增效率。
(2)荧光PCR检测
通过检测新型冠状病毒核酸检测试剂国家参考品(批号370099-202001),可以测试各组引物探针是否存在交叉反应,具体数据见实施例3中的准确度检测,结果结果表明39种引物探针特异性很好,相互之间没有交叉反应。
实施例2特异性检测
在上海宏石医疗科技有限公司Slan-96型号PCR仪上,检测10份健康志愿者咽拭子标本假病毒阳性对照和DEPC处理水,检测结果如下:
如表1、图2所示,检测结果表明,本发明的引物探针组合特异性正常,与健康人和常见其他呼吸道病原体没有非特异性扩增,试剂盒特异性较好。
实施例3准确度检测
在上海宏石医疗科技有限公司Slan-96型号PCR仪上,检测新型冠状病毒核酸检测试剂国家参考品(批号370099-202001)。按照上述标本处理方法处理参考品中培养物,检测结果见表2,图3-8,
上述检测结果表明,本发明的引物探针组合检测新型冠状病毒核酸检测试剂国家参考品,具有很高的准确度,与阳性符合率100%。
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.呼吸道症候群病原体核酸检测试剂盒,其特征在于,所述试剂盒用于检测39种呼吸道病原体和人源内参基因GAPDH,共计40个靶基因,采用5色荧光PCR技术,使用整条8连管,每管中包括检测5种靶基因的特异性引物和探针,8连管的组合如下:
1管:冠状病毒SARS-CoV、冠状病毒HCoV-NL63、冠状病毒HCoV-HKU1、冠状病毒229E、冠状病毒HCoV-OC43;
2管:冠状病毒MERS-CoV、新型冠状病毒SARS-CoV-2、副流感病毒1型、副流感病毒2型、副流感病毒3型;
3管:甲型流感病毒H1N1、甲型流感病毒H5N1、甲型流感病毒H7N9、乙型流感病毒Victoria、乙型流感病毒Yamagata;
4管:呼吸道合胞疱疹病毒A型、呼吸道合胞疱疹病毒B型、鼻病毒、人偏肺病毒、丙型流感病毒;
5管:肠道病毒CA16、肠道病毒EV71、人巨细胞病毒、腺病毒、EB病毒;
6管:肺炎支原体、肺炎衣原体、沙眼衣原体、鹦鹉热衣原体、Q热立克次体;
7管:肺炎链球菌、肺炎克雷伯菌、金黄色葡萄球菌、铜绿假单胞菌、化脓性链球菌;
8管:耐甲氧西林葡萄球菌、鲍曼不动杆菌、嗜肺军团菌、流感嗜血杆菌、人源内参基因GAPDH;
其中,所述特异性引物为,
新型冠状病毒SARS-Cov-2的引物序列:
SEQ ID NO. 1.:5'-TTCCATGTGGGCTCTTATAATC-3',
SEQ ID NO. 2:5'-TCAACACACATAAAAACAATACCTC-3';
冠状病毒HCoV-229E的引物序列:
SEQ ID NO. 3:5'-GTTTTCATCTTCATTCTCTGTTGC-3';
SEQ ID NO. 4:5'-TGTCACAAGAAAACAACAAAAAAT-3';
冠状病毒SARS-CoV的引物序列:
SEQ ID NO. 5:5'-GAGTAAAATTTTGGCTTATGTCA-3';
SEQ ID NO. 6:5'-CAATAGTTGTAGGTAGTGAAGCTCT-3';
冠状病毒HCoV-OC43的引物序列:
SEQ ID NO. 7:5'-CTTGTGCTGGAAGGCGT-3';
SEQ ID NO. 8:5'-TTCAAACACTCCACATGCAG-3';
冠状病毒MERS-CoV的引物序列:
SEQ ID NO. 9:5'-GTCCAAAAAGAGTCAGATGAGTAT-3';
SEQ ID NO. 10:5'-ACGACATGCAGGATATTCTTAG-3';
冠状病毒HCoV-HKU1的引物序列:
SEQ ID NO. 11:5'-GTGGTTATCTTATAAGGTGGGTTA-3';
SEQ ID NO. 12:5'-TACGCATTAATATCAAACTGGTT-3';
冠状病毒HCoV-NL63的引物序列:
SEQ ID NO. 13:5'-TTGGGTGTTCGTGTTTTAGAT-3';
SEQ ID NO. 14:5'-CTCAATAGAATCATCCAAAAGC-3';
甲型H1N1流感病毒的引物序列:
SEQ ID NO. 15:5'-GCCCCCTCAAAGCCGA-3';
SEQ ID NO. 16:5'-CGCTCACTGGGCACGG-3';
甲型H5N1流感病毒的引物序列:
SEQ ID NO. 17:5'-GCCCCCTCAAAGCCGA-3';
SEQ ID NO. 18:5'-CGCTCACTGGGCACGG-3';
甲型H7N9流感病毒的引物序列:
SEQ ID NO. 19:5'-GCCCCCTCAAAGCCGA-3';
SEQ ID NO. 20:5'-CGCTCACTGGGCACGG-3';
乙型流感病毒Victoria型的引物序列:
SEQ ID NO. 21:5'-TTGCAAATCTCAAAGGAACA-3'
SEQ ID NO. 22:5'-TGCATTATAGGAAAGCACCC-3'
乙型流感病毒Yamagata型的引物序列:
SEQ ID NO. 23:5'-TTGCAAATCTCAAAGGAACA-3';
SEQ ID NO. 24:5'-TGCATTATAGGAAAGCACCC-3';
丙型流感病毒的引物序列:
SEQ ID NO. 25:5'-GACGACTACACACCAGACATCC-3';
SEQ ID NO. 26:5'-AATTTTTAAAGCTGTTCTTCCAC-3';
副流感病毒1型的引物序列:
SEQ ID NO. 27:5'-ACAATCAAAGAGACAATCACAGAA-3';
SEQ ID NO. 28:5'-GATCTCTGCTTTGCTTGTTTAAC-3';
副流感病毒2型的引物序列:
SEQ ID NO. 29:5'-CTAAGTGATGGAATCAATCGC-3';
SEQ ID NO. 30:5'-AGGTCAGTTGTGGCATAATCTT-3';
副流感病毒3型的引物序列:
SEQ ID NO. 31:5'-AAGTCATGTTCTCTAGCACTCCT-3';
SEQ ID NO. 32:5'-TTGTTGAGATTGAGCCATCATA-3';
呼吸道合胞疱疹病毒A 型的引物序列:
SEQ ID NO. 33:5'-GTYTTAACCAGCAAAGTGTTAGA-3';
SEQ ID NO. 34:5'-CACCTGCATTRACACTAAATTCT-3';
呼吸道合胞疱疹病毒B型的引物序列:
SEQ ID NO. 35:5'-GTYTTAACCAGCAAAGTGTTAGA-3';
SEQ ID NO. 36:5'-CACCTGCATTRACACTAAATTCT-3';
鼻病毒HRV的引物序列:
SEQ ID NO. 37:5'-TTGAGTCCTCCGGCCC-3';
SEQ ID NO. 38:5'-AAACACGGACACCCAAAGTA-3';
人偏肺病毒的引物序列:
SEQ ID NO. 39:5'-YGACARTCAARATGATTATGAGTAA-3';
SEQ ID NO. 40:5'-GCTGCTTCATTACCCATGAA-3';
肠道病毒EV71的引物序列:
SEQ ID NO. 41:5'-CGTTCTCAGTGCGGACTG-3';
SEQ ID NO. 42:5'-TAGGTAGTTCTGGTTRCGCATY-3';
肠道病毒CA16的引物序列:
SEQ ID NO. 43:5'-GGCGGAAATGCGAGTT-3';
SEQ ID NO. 44:5'-GTACTGYAGYAATTGGGGGA-3';
人巨细胞病毒HCMV的引物序列:
SEQ ID NO. 45:5'-AGAGGAGACTAGTGTGATGCTG-3';
SEQ ID NO. 46:5'-AATGTACTGGGCAAAGACCT-3';
腺病毒AD的引物序列:
SEQ ID NO. 47:5'-GACATGACYTTYGAGGTGGA-3';
SEQ ID NO. 48:5'-GTAGACGGCCTCGATGA-3';
EB病毒的引物序列:
SEQ ID NO. 49:5'-CTAACTCCAACGAGGGCAG-3';
SEQ ID NO. 50:5'-GGACCACCTCCAGGTGC-3';
肺炎支原体MP的引物序列:
SEQ ID NO. 51:5'-ACCAATGCCATCAACCC-3';
SEQ ID NO. 52:5'-AGTGATGCCCTTAGCGC-3';
肺炎衣原体CP的引物序列:
SEQ ID NO. 53: 5'-TTCCCTTGCCAACAGACG-3';
SEQ ID NO. 54:5'-GGCTGAGCAATGCGGATGT-3';
沙眼衣原体CT的引物序列:
SEQ ID NO. 55:5'-AATGCAAATCGTTTCCTTG-3';
SEQ ID NO. 56:5'-TTGTGCATTTACGTGAGCTG-3';
鹦鹉热衣原体的引物序列:
SEQ ID NO. 57:5'-TACGGGTTCCGCTCTCTC-3';
SEQ ID NO. 58:5'-GCGTCACACCAAGTAGMG-3';
Q热立克次体的引物序列:
SEQ ID NO. 59:5'-AATACGGACTCACGATGGC-3';
SEQ ID NO. 60:5'-AACACCCGTTTCATCCG-3';
肺炎链球菌SA的引物序列:
SEQ ID NO. 61:5'-ATTGTTGGGAACGGTTGC-3';
SEQ ID NO. 62:5'-TTTCAATCAGTTCAACCGCT-3';
肺炎克雷伯菌KPN的引物序列:
SEQ ID NO. 63:5'-GACACGGTCCAGACTCCTAC-3';
SEQ ID NO. 64:5'-AAGTGCTTTACAACCCGAA-3';
金黄色葡萄球菌的引物序列:
SEQ ID NO. 65:5'-GAGTTAGATAAATATTTACAGCAACA-3';
SEQ ID NO. 66:5'-GGCACGATATCTTTATCATATAGA-3';
铜绿假单胞菌PA的引物序列:
SEQ ID NO. 67:5'-GACCGCTACCGAAGACG-3';
SEQ ID NO. 68:5'-CGCTCGTTAGCCTCGTC-3';
化脓性链球菌的引物序列:
SEQ ID NO. 69:5'-ACCTACTTATAGCGGAAGAGAAT-3';
SEQ ID NO. 70:5'-ACGAGAGCTACCTGCAGAAC-3';
耐甲氧西林葡萄球菌的引物序列:
SEQ ID NO. 71:5'-AGTTAGATTGGGATCATAGCGT-3';
SEQ ID NO. 72:5'-TTCCACATTGTTTCGGTCTAA-3';
鲍曼不动杆菌的引物序列:
SEQ ID NO. 73:5'-ACACACTACGGGTGTTTTAGTT-3';
SEQ ID NO. 74:5'-AGCATTAAGCATTTTGAAGGT-3';
嗜肺军团菌LP的引物序列:
SEQ ID NO. 75:5'-ACATCATTAGCTACAGACAAGGA-3';
SEQ ID NO. 76:5'-TCGGTTAAAGCCAATTGAG-3';
流感嗜血杆菌Hi的引物序列:
SEQ ID NO. 77:5'-GTCGTTTGTATGATGTTGATCC-3';
SEQ ID NO. 78:5'-CGGGCTTTCATCCCTG-3';
人源内参基因GAPDH基因的引物序列:
SEQ ID NO. 118:5'-ATGGACACGCTCCCCTGAC-3';
SEQ ID NO. 119:5'-TGAGTCCTTCCACGATACCAAA-3';
探针序列为:
新型冠状病毒SARS-Cov-2的探针序列:
SEQ ID NO. 79:5'-TGTTACTTCTAACTACTCAGGTG-3';
冠状病毒HCoV-229E的探针序列:
SEQ ID NO. 80:5'-CTTAATTGTGCATTAGGTGC-3';
冠状病毒SARS-CoV的探针序列:
SEQ ID NO. 81:5'-ATTCTTAGGACAAGCAGC-3';
冠状病毒HCoV-OC43的探针序列:
SEQ ID NO. 82:5'-AAGGAACAGCCTACAGTAA-3';
冠状病毒MERS-CoV的探针序列:
SEQ ID NO. 83:5'-TTCTGGCTAAAGGGC-3';
冠状病毒HCoV-HKU1的探针序列:
SEQ ID NO. 84:5'-CCACTATTCCTAAAGC-3';
冠状病毒HCoV-NL63的探针序列:
SEQ ID NO. 85:5'-TCTGATAATAATTGTTGGATT-3';
甲型H1N1流感病毒的探针序列:
SEQ ID NO. 86:5'-TCTCATGGAATGGCTAAAGACAA-3';
甲型H5N1流感病毒的探针序列:
SEQ ID NO. 87:5'-TCTCATGGAGTGGCTAAAGACAA-3';
甲型H7N9流感病毒的探针序列:
SEQ ID NO. 88:5'-CATGGAGTGGATAAAGACAAGACC-3';
乙型流感病毒Victoria型的探针序列:
SEQ ID NO. 89:5'-CTTGCCGAGGGTATTTTCC-3';
乙型流感病毒Yamagata型的探针序列:
SEQ ID NO. 90:5'-TTAGCAGAAGGTGTGGTCCC-3';
丙型流感病毒的探针序列:
SEQ ID NO. 91:5'-CAAGAAAAGTGAAAGATACAGGAG-3';
副流感病毒1型的探针序列:
SEQ ID NO. 92:5'-AGAAGTGATATCAAGGAC-3';
副流感病毒2型的探针序列:
SEQ ID NO. 93:5'-TGCTATACCAGGAGGTTGTGTC-3';
副流感病毒3型的探针序列:
SEQ ID NO. 94:5'-TCAAGTACAATATCTTCTA-3';
呼吸道合胞疱疹病毒A 型的探针序列:
SEQ ID NO. 95:5'-AGATAAACAGTTGTTACCTA-3';
呼吸道合胞疱疹病毒B型的探针序列:
SEQ ID NO. 96:5'-TTCCAACAATCTGCTGTT-3';
鼻病毒HRV的探针序列:
SEQ ID NO. 97:5'-GGCTAACCYTAACCCT-3';
人偏肺病毒的探针序列:
SEQ ID NO. 98:5'-TGGGACAAGTCAAAAT-3';
肠道病毒EV71的探针序列:
SEQ ID NO. 99:5'-TCCAAGTCCAAGTACCCTTTAG-3';
肠道病毒CA16的探针序列:
SEQ ID NO. 100:5'-ATGCGCTTTGATGC-3';
人巨细胞病毒HCMV的探针序列:
SEQ ID NO. 101:5'-ATAACCAAGCCTGAGG-3';
腺病毒AD的探针序列:
SEQ ID NO. 102:5'-CCCATGGATGAGCCCAC-3';
EB病毒的探针序列:
SEQ ID NO. 103:5'-TTGAGAGCAGAGTGGG-3';
肺炎支原体MP的探针序列:
SEQ ID NO. 104:5'-TTAACCCCGTGAACG-3';
肺炎衣原体CP的探针序列:
SEQ ID NO. 105:5'-CGACCATCAATTATC-3';
沙眼衣原体CT的探针序列:
SEQ ID NO. 106:5'-AAATCTAGAAAATCTTGCG-3';
鹦鹉热衣原体的探针序列:
SEQ ID NO. 107:5'-CAAGCCTTGCCTGTAG-3';
Q热立克次体的探针序列:
SEQ ID NO. 108:5'-CCAAGCGATTTTATGA-3';
肺炎链球菌SA的探针序列:
SEQ ID NO. 109:5'-TAGGACCTGTTGATAATG-3';
肺炎克雷伯菌KPN的探针序列:
SEQ ID NO. 110:5'-AGCAGTGGGGAATAT-3';
金黄色葡萄球菌的探针序列:
SEQ ID NO. 111:5' AATTAGATCCGTATTGGTTA-3';
铜绿假单胞菌PA的探针序列:
SEQ ID NO. 112:5'-GCTCGTGCTCAGGCTCG-3';
化脓性链球菌的探针序列:
SEQ ID NO. 113:5'-ATTTCAGAATTGATGGCT-3';
耐甲氧西林葡萄球菌的探针序列:
SEQ ID NO. 114:5'-AATGCAGAAAGACCAAA-3';
鲍曼不动杆菌的探针序列:
SEQ ID NO. 115:5'-AACTCAACAAAGCTATG-3';
嗜肺军团菌LP的探针序列:
SEQ ID NO. 116:5'-ATAGCATTGGTGCCGATTT-3';
流感嗜血杆菌Hi的探针序列:
SEQ ID NO. 117:5'-ACTCGTTTTACAAAAGA-3';
人源内参基因GAPDH基因的探针序列:
SEQ ID NO. 120:5'-GCAATGCCTCCTGCACCACCAA-3';
所述探针的5’端标记荧光基团,3’端标记淬灭基团;
所述荧光基团为FAM、HEX、JOE、TET、CY3、CY5、ROX或Texas;
所述淬灭基团为TAMRA、BHQ或MGB。
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