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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2024, Vol. 44 Issue (4): 43-53    DOI: 10.13523/j.cb.2307023
技术与方法     
恙虫病东方体超快速检测方法的建立及评价
金捷1*,陆洪斌2*,张怡3,王新宇3,吴晶3,李涛3,**(),姜宁1,**()
1 复旦大学生命科学学院 上海 200438
2 云南省红河州金平县人民医院 红河 661599
3 复旦大学附属华山医院 上海 200040
Establishment and Evaluation of an Ultra-rapid Method for the Detection of Orientia tsutsugamushi
JIN Jie1*,LU Hongbin2*,ZHANG Yi3,WANG Xinyu3,WU Jing3,LI Tao3,**(),JIANG Ning1,**()
1 School of Life Sciences, Fudan University, Shanghai 200438, China
2 The People’s Hospital of Jinping County in Honghe Prefecture of Yunnan Province, Honghe 661599, China
3 Huashan Hospital Affiliated to Fudan University, Shanghai 200040, China
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摘要：

目的:建立一种可以在基层医疗机构使用的超快速恙虫病东方体(Orientia tsutsugamushi,Ot)核酸检测方法。方法:利用实时荧光定量PCR技术(quantitative real-time polymerase chain reaction,qPCR),通过比对分析数据库中恙虫病东方体全基因组序列确定靶基因,设计qPCR特异性引物和探针,优化反应体系和反应程序,建立超快速多重qPCR检测方法;利用经过数字PCR(droplet digital PCR,ddPCR)定量后的样本核酸作为标准物质,评价该方法的灵敏度、重复性以及最低检出限;通过比较基因组学分析,验证恙虫病东方体与同科属的易引起类似症状的病原体的特异性,通过检测24种(共45例)易引起类似症状的其他病原体核酸评价特异性;利用116例来源于云南省红河州金平县人民医院和复旦大学附属华山医院的临床全血样本,通过四格表卡方检验对多重qPCR超快速检测方法和传统qPCR检测方法的检测结果进行比较,采用Kappa一致性检验分析方法间的一致性。结果:恙虫病东方体与同科属的易引起类似症状的病原体的同源性较低,与常见的易引起类似症状的24种病原体无交叉反应,具有很好的特异性;最低检出限为156 copies/mL;具有很好的重复性,检测Ct值的变异系数≤3.13%;多重qPCR超快速检测方法和传统qPCR检测方法同时检测116例临床全血样本,检测结果的差异无统计学意义(P>0.05),两种方法具有很高的一致性(Kappa=0.939 2,P<0.001),总符合率97.42%(CI:92.67%~99.12%),检测时间可缩短至30 min。结论:建立的多重qPCR超快速检测方法具有良好的灵敏度、重复性和特异性,可以快速诊断临床全血样本中的恙虫病东方体核酸,在基层医疗机构实现及时快速诊疗。
关键词： 恙虫病;  恙虫病东方体;  多重qPCR技术;  超快速核酸检测    
Abstract:

Objective: To establish an ultra-fast nucleic acid test for Orientia tsutsugamushi that can be used in grassroots medical institutions. Methods: Using quantitative real-time PCR (qPCR), we compared and analyzed the whole genome sequences of Orientia tsutsugamushi in the database, identified the target genes, designed qPCR-specific primers and probes, optimized the reaction system and reaction procedure, and established a multiplex qPCR ultra-rapid assay. The sensitivity, reproducibility, and minimum detection limit of the method were evaluated using viral nucleic acids quantified by droplet digital PCR (ddPCR) as standards. Comparative genomic analysis was performed to verify the specificity of Orientia tsutsugamushi with pathogens of the same family or genus that tend to cause similar symptoms. Specificity was evaluated using nucleic acids from 24 (45 cases in total) other pathogens that tend to cause similar symptoms. The results of the multiplex qPCR ultra-rapid assay and the conventional qPCR assay were compared using 116 clinical whole blood samples by four-grid table chi-square test, and the agreement between methods was analyzed using the Kappa consistency test. Results: Orientia tsutsugamushi has low homology to pathogens of the same family or genus that tend to cause similar symptoms. Multiplex qPCR ultra-rapid assay showed good specificity and no cross-reactivity with 24 common pathogens. The minimum detection limit was 156 copies/mL. Repeatability comparison analysis showed a coefficient of variation of ≤ 3.13% for Ct values for each concentration assay. The differences between the multiplex qPCR ultra-rapid assay and the conventional qPCR assay for simultaneous testing of 116 clinical whole blood samples were not statistically significant (P>0.05), the two methods had high agreement (Kappa=0.939 2,P<0.001),with total compliance rate 97.42% (CI: 92.67%~99.12%), and the testing time could be be reduced to 30 min. Conclusion: The multiplex qPCR ultra-rapid assay has good sensitivity, reproducibility and specificity, which can rapidly diagnose Orientia tsutsugamushi nucleic acid in clinical whole blood samples and achieve timely and rapid treatment in grassroots medical institutions.
Key words: Tsutsugamushi disease    Orientia tsutsugamushi    qPCR Multiplex    qPCR ultra-rapid assay
收稿日期: 2023-07-17 出版日期: 2024-04-30
ZTFLH:  Q81  
通讯作者: ** 电子信箱:litaokc@126.com;ningjiang@fudan.edu.cn   
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金捷
陆洪斌
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李涛
姜宁

引用本文:

金捷, 陆洪斌, 张怡, 王新宇, 吴晶, 李涛, 姜宁. 恙虫病东方体超快速检测方法的建立及评价[J]. 中国生物工程杂志, 2024, 44(4): 43-53.

JIN Jie, LU Hongbin, ZHANG Yi, WANG Xinyu, WU Jing, LI Tao, JIANG Ning. Establishment and Evaluation of an Ultra-rapid Method for the Detection of Orientia tsutsugamushi. China Biotechnology, 2024, 44(4): 43-53.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2307023    或    https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I4/43

特异性样本 性质 来源
甲型流感病毒(H1N1、H3N2)、乙型流感病毒、呼吸道合胞病毒 咽拭子样本 上海市公共卫生临床中心
新型冠状病毒 咽拭子样本 上海思路迪医学检验所有限公司
呼吸道合胞病毒、肺炎支原体、腺病毒、鼻病毒、肠道病毒 鼻咽拭子样本 珠海市人民医院
甲型流感病毒(2009H1N1-01、H1N1-05、H3N2-03)、乙型流感病毒(BY-04、BV-02)、腺病毒(ADV7-01)、人偏肺病毒(HMPV-29、HMPV-10) 标准株 广州斯坦达生物科技有限公司
人巨细胞病毒(ATCCVR-538)、轮状病毒(ATCC-VR2104)、诺如病毒(ATCCVR-3234SD)、腮腺炎病毒(ATCCVR-106)、水痘-带状疱疹病毒(ATCCVR-1832)、肺炎衣原体(ATCCVR-2282) 标准株 American Type Culture Collection(ATCC)
军团菌(BNCC337413)、百日咳杆菌(BNCC337541)、流感嗜血杆菌(BNCC289143)、金黄色葡萄球菌(BNCC186335)、肺炎链球菌(BNCC338425)、肺炎克雷伯菌(BNCC186113) 标准株 商城北纳创联生物科技有限公司
化脓性链球菌(101010127)、结核分枝杆菌(BW-027) 病毒核酸 商城北纳创联生物科技有限公司
表1  用于特异性检测的样本信息
基因 编号 上游引物 下游引物 探针 通道设置
groEL groEL1 G-F1 G-R1 G-P1 Cy5
groEL2 G-F1 G-R2 G-P1 Cy5
tsa56 tsa56-1 56-F1 56-R1 56-P1 FAM
tsa56-2 56-F1/56-F2 56-R1 56-P1 FAM
tsa56-3 56-F1 56-R2/56-R3 56-P1 FAM
tsa56-4 56-F1/56-F2 56-R3/56-R4 56-P1 FAM
TraD TraD1 T-F1 T-R1 T-P1 ROX
TraD2 T-F2 T-R2 T-P2 ROX
ERV3-1(内标) RnaseP RnaseP-F RnaseP-R RnaseP-P VIC
GAPDH GAPDH-F GAPDH-R GAPDH-P VIC
ERV3-1 ERV-F ERV-R ERV-P VIC
表2  多重qPCR引物和探针筛选结果
目的基因 引物/探针 序列(5' → 3') 5'修饰 3'修饰 Tm值 序列长度/bp
groEL 上游引物 GCCCAATTTGTTATATCAGTTGCTAGTA - - 59.0 94
下游引物 CCTAACTGCAGCATCAGCTATAACTG - - 59.5 -
荧光探针 AGCTGATGTAGCTGGTGATGGTACAACTACTGC 5'Cy5 3'BHQ3 68.3 -
tsa56 上游引物 ATTCCTCCAACRAYTCCAACTTTA - - 57.6 107
下游引物1 TGTCTGCGTTGTCGTTGCC - - 60.4 -
下游引物2 CAATGTCTGCATTGTCATTGCC - - 60.0 -
荧光探针 TAAGGACCACACTCTAAT 5'FAM 3'MGB 68.0 -
TraD 上游引物 CGCTAAAAAGGCCTCGAAAAT - - 58.6 109
下游引物 CATATTAGTTTTACCAGTACCTGTTGTTCC - - 58.8 -
荧光探针 TTGGAGGCTTGCCATTAGTAAAGAATAGTGAAAG 5'ROX 3'BHQ2 67.0 -
ERV3-1(内标) 上游引物 CATGGGAAGCAAGGGAACTAATG - - 60.6 135
下游引物 CCCAGCGAGCAATACAGAATTT - - 59.2 -
荧光探针 TCTTCCCTCGAACCTGCACCATCAAGTCA 5'VIC 3'BHQ1 72.3 -
表3  多重qPCR引物和探针序列
优化的组分名称 组C1终浓度 组C2终浓度 组C3终浓度 组C4终浓度
MgCl2 3 mmol/L 4 mmol/L 5 mmol/L 6 mmol/L
dNTPs 0.1 mmol/L 0.2 mmol/L 0.3 mmol/L 0.4 mmol/L
Tris(pH 8.8) 0 mol/L 0.01 mol/L - -
(NH4)2SO4 0 mol/L 0.01 mol/L - -
Taq DNA聚合酶 2.5 U 5 U 10 U -
BSA 0% 0.16% - -
表4  多重qPCR反应体系优化
图1  多重qPCR超快速检测方法的标准曲线
图2  多重qPCR超快速检测方法的特异性验证 A:比较基因组学分析 恙虫病东方体(Orientia tsutsugamushi)、中东东方体(Orientia chuto)、普氏立克次体(Rickettsia prowazekii)、贝纳特氏立克次体(Coxiella burnetii)、查菲埃立克体(Ehrlichia chaffeensis)比较基因组学圈图 B:groEL基因 C:tsa56基因 D:TraD基因
groEL基因 tsa56基因 TraD基因
浓度(copies/mL) 变异系数 检出率/% 浓度(copies/mL) 变异系数 检出率/% 浓度(copies/mL) 变异系数 检出率/%
5 000 0.31% (5/5) 100 5 000 0.80% (5/5) 100 5 000 1.84% (3/3) 100
2 500 0.74% (5/5) 100 2 500 1.10% (5/5) 100 2 848 0.84% (10/10) 100
1 250 1.80% (10/10) 100 1 250 1.01% (10/10) 100 2 500 1.13% (3/3) 100
625 1.62% (10/10) 100 625 3.13% (10/10) 100 1 424 0.80% (10/10) 100
313 2.24% (10/10) 100 313 (8/10) 80 1 250 0.18% (3/3) 100
156 2.02% (10/10) 100 156 (8/10) 80 712 1.55% (10/10) 100
78 (5/10) 50 78 (4/10) 40 625 0.26% (3/3) 100
39 (5/10) 50 39 (4/10) 40 356 (8/10) 80
20 (2/10) 20 20 (0/10) 0 313 (3/6) 50
10 (1/10) 10 10 (1/10) 10 178 (7/10) 70
- - - - - - 156 (3/6) 50
表5  多重qPCR超快速检测方法的检出限和重复性验证
多重qPCR超快速
检测方法		 传统qPCR检测方法
阳性/例 阴性/例 合计/例
阳性 79 3 82
阴性 0 34 34
合计 79 37 116
表6  多重qPCR超快速检测方法的临床验证
统计指标 多重qPCR超快速检测方法
阳性符合率 100.00% (CI:95.36%~100.00%)
阴性符合率 91.89% (CI:78.70%~97.20%)
总符合率 97.42% (CI:92.67%~99.12%)
卡方检验P值 0.250
Kappa值 0.939 2 (CI:0.871 3~1.000 0)
Kappa一致性检验P值 0.000
表7  多重qPCR超快速检测方法的临床统计分析
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