液相芯片技术在感染性腹泻病原体检测中应用

曹军; 管红霞

doi:10.11847/zgggws1133735

液相芯片技术在感染性腹泻病原体检测中应用

doi: 10.11847/zgggws1133735

曹军¹,
管红霞^2, ,

1.
无锡市惠山区疾病预防控制中心, 江苏无锡 214100
2.
无锡市疾病预防控制中心

基金项目: 无锡市卫健委项目（T202017）；无锡市科技局项目（锡科社[2020]259号 – 24）

详细信息

作者简介:
曹军（1976 – ），男，江苏无锡人，副主任技师，本科，研究方向：传染病病原监测

通讯作者:
管红霞，E-mail：ghx-331@163.com

中图分类号: R 115
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- 文章访问数: 298
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- 被引次数: 0
出版历程
- 接收日期: 2020-12-29
- 网络出版日期: 2022-03-23
- 刊出日期: 2022-07-01

Application of liquid-phase chip technology in infectious diarrhea pathogen detection

CAO Jun¹,
GUAN Hong-xia^{2
, ,}

1.
Huishan District Center for Disease Control and Prevention, Wuxi, Jiangsu Province 214100, China

摘要

摘要: 目的评价液相芯片技术检测感染性腹泻病原体的应用价值。方法于2015年4月 — 2016年4月收集江苏省无锡市人民医院（含儿童医院）肠道门诊就诊的179份腹泻病例粪便标本，采用美国Luminex公司胃肠道病原体检测试剂盒（xTAG GPP)进行检测，并与real-time PCR检测结果进行比较。结果 xTAG GPP检出9种病原体82例，阳性检出率为45.81 %（82/179），real-time PCR检出9种病原体81例，阳性检出率为45.25 %（81/179），两种检测方法总符合率92.17 %（165/179）。xTAG GPP检测腹泻病原体灵敏度为84.44 %，特异度为93.33 %，阳性预测值为92.68 %，阴性预测值为85.71 %。结论与传统的检测方法相比，液相芯片技术检测通量高、速度快，对常见腹泻病原体具有较高的敏感性和特异性，可用于腹泻病的快速诊断。
- 液相芯片技术 /
- 感染性腹泻 /
- real-time PCR
Abstract: Objective To evaluate application value of liquid phase chip technology in the detection of infectious diarrhea pathogens. Methods From April 2015 through April 2016, 179 stool samples of diarrhea cases treated in the intestinal outpatient department of Wuxi People's Hospital (including Children′s Hospital) of Jiangsu province were collected and detected with xTAG® Gastrointestinal Pathogen Panel (xTAG GPP), and the detection results were compared to those with real-time PCR. Results Totally 9 pathogens were detected in the samples from 82 diarrhea cases with xTAG GPP, with a positive detection rate of 45.81% (82/179); while, 9 pathogens were detected in 81 cases with real-time PCR, with a positive detection rate of 45.25% (81/179). The total coincidence rate for the two detections was 92.17% (165/179). The sensitivity and specificity of xTAG GPP for diarrhea pathogen detection were 84.44% and 93.33%, and positive and negative predictive value were 92.68% and 85.71%, respectively. Conclusion Compared with real-time PCR detection, liquid phase chip technology is of high throughput, speed, sensitivity and specificity in common diarrhea pathogen detections, and could be used for rapid pathogenic diagnosis of diarrhea diseases.
- liquid phase chip technology /
- infectious diarrhea /
- real-time PCR

HTML全文

表 1 xTAG GPP和real-time PCR检测病原及目标基因

xTAG GPP		目标基因
病原中文名称	病原英文名称	real-time PCR	TPT real-time PCR
腺病毒40/41型	Adenovirus 40/41	hexon（内部使用）	hexon ^[3-4]
诺如病毒GI/GII	Norovirus GI/GII	ORF1-ORF2 junction（内部使用）	RDRP of GI ^[5]; RDRP and ORF1-ORF2 junction of GII ^a^[6-7]
A组轮状病毒	Rotavirus A	NSP3（内部使用）	VP6 ^[7]
弯曲菌	Campylobacter	C. jejuni hipO; C. coli glyA（内部使用）	glyA of C. jejuni and C. coli ^[8]，cadF ^[9]
艰难梭菌（毒素A/B型）	C. difficile toxin A/B	tcdA，tcdB（内部使用）	tcdA，tcdB ^[10-12]
肠出血性大肠埃希菌O157	E. coli O157
肠产毒性大肠埃希菌LT/ST型	ETEC LT/ST	elt，estA（STh），estB（STp）（内部使用）	elt，estA（STh），estB（STp）^[13]
产志贺毒素大肠埃希菌stx1/stx2型	STEC stx1/stx2	stx1，stx2（内部使用）	stx1，stx2 ^[13]
肠侵袭性大肠埃希菌/志贺菌	EIEC/Shigella	ipaH（内部使用）	virA ^[14]
沙门菌	Salmonella	ttrRSBCA（内部使用），培养法	ompC ^[15]
霍乱弧菌	V. cholerae	ompW（内部使用）	epsM ^[16]
小肠结肠炎耶尔森氏菌	Y. enterocolitica	ystA（内部使用）	ail ^[17]
隐孢子虫	Cryptosporidium	18S rRNA gene（内部使用）	18S rRNA gene ^[18-19]
痢疾阿米巴	E. histolytica	SSU rRNA gene（内部使用）	SSU rRNA gene ^[20-21]
蓝氏贾第鞭毛虫	G. lamblia	SSU RNA gene（内部使用）	hsp^[18]
注：a 使用两对引物对诺如病毒GII进行不一致分析，任何一对引物为阳性，则判定标本为阳性。

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表 2 不同方法病原体的检测结果

病原体	培养法	xTAG GPP		real-time PCR		总阳性数 ^a	总阳性率（%）
病原体	阳性数	阳性数	阳性率（%）	阳性数	阳性率（%）	总阳性数 ^a	总阳性率（%）
诺如病毒GI/GII型（Norovirus GI/GII）		25	13.97	25	13.97	25	13.97
A组轮状病毒（Rotavirus A）		23	12.85	23	12.85	24	12.85
腺病毒40/41型（Adenovirus 40/41）		3	1.68	4	2.23	4	2.23
沙门菌（Salmonella）	17	5	2.79	8	4.47	17	9.50
弯曲菌（Campylobacter）		9	5.03	10	5.59	10	5.59
肠产毒性大肠埃希菌（ETEC）		2	1.12	2	1.12	2	1.12
艰难梭菌（毒素A/B）（C. difficile toxin A/B）		4	2.23	4	2.23	4	2.23
志贺菌（Shigella）		8	4.47	6	3.35	6	3.35
霍乱弧菌（V. cholerae）		2	1.12	2	1.12	2	1.12
合计 ^b	17	82	45.81	81	45.25	90	50.28
注：a 阳性数为培养法、xTAG GPP和real-time PCR检测为阳性标本的总和；b 因部分标本同时感染两种或多种病原体，不计算此类靶标在标本中的阳性检出率。

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表 3 xTAG GPP和real-time PCR检测不同病原体检测的灵敏度和特异度

病原体	金标准^a	xTAG GPP		灵敏度（%）	特异度（%）	阳性预测值（%）	阴性预测值（%）	real-time PCR		灵敏度（%）	特异度（%）	阳性预测值（%）	阴性预测值（%）
病原体	金标准^a	P	N	灵敏度（%）	特异度（%）	阳性预测值（%）	阴性预测值（%）	P	N	灵敏度（%）	特异度（%）	阳性预测值（%）	阴性预测值（%）
Norovirus GI	P	6	0	100.00	100.00	100.00	100.00	6	0	100.00	100.00	100.00	100.00
Norovirus GI	N	0	173	100.00	100.00	100.00	100.00	0	173	100.00	100.00	100.00	100.00
Norovirus GII	P	20	0	100.00	99.37	95.24	100.00	21	0	100.00	100.00	100.00	100.00
Norovirus GII	N	1	158	100.00	99.37	95.24	100.00	0	158	100.00	100.00	100.00	100.00
Rotavirus A	P	23	0	100.00	100.00	100.00	100.00	23	0	100.00	100.00	100.00	100.00
Rotavirus A	N	0	156	100.00	100.00	100.00	100.00	0	156	100.00	100.00	100.00	100.00
Adenovirus 40/41	P	3	1	75.00	100.00	100.00	99.43	4	0	100.00	100.00	100.00	100.00
Adenovirus 40/41	N	0	175	75.00	100.00	100.00	99.43	0	175	100.00	100.00	100.00	100.00
Salmonella	P	5	12	29.41	100.00	100.00	93.10	8	9	47.06	100.00	100.00	94.74
Salmonella	N	0	162	29.41	100.00	100.00	93.10	0	162	47.06	100.00	100.00	94.74
Campylobacter	P	9	1	90.00	100.00	100.00	99.41	10	0	100.00	100.00	100.00	100.00
Campylobacter	N	0	169	90.00	100.00	100.00	99.41	0	169	100.00	100.00	100.00	100.00
ETEC（LT）	P	1	0	100.00	100.00	100.00	100.00	1	0	100.00	100.00	100.00	100.00
ETEC（LT）	N	0	178	100.00	100.00	100.00	100.00	0	178	100.00	100.00	100.00	100.00
ETEC（ST）	P	1	0	100.00	100.00	100.00	100.00	1	0	100.00	100.00	100.00	100.00
ETEC（ST）	N	0	178	100.00	100.00	100.00	100.00	0	178	100.00	100.00	100.00	100.00
C. difficile toxin A/B	P	4	0	100.00	100.00	100.00	100.00	4	0	100.00	100.00	100.00	100.00
C. difficile toxin A/B	N	0	175	100.00	100.00	100.00	100.00	0	175	100.00	100.00	100.00	100.00
Shigella	P	6	0	100.00	98.84	75.00	100.00	6	0	100.00	100.00	100.00	100.00
Shigella	N	2	171	100.00	98.84	75.00	100.00	0	173	100.00	100.00	100.00	100.00
V. cholerae	P	2	0	100.00	100.00	100.00	100.00	2	0	100.00	100.00	100.00	100.00
V. cholerae	N	0	177	100.00	100.00	100.00	100.00	0	177	100.00	100.00	100.00	100.00
合计	P	76	14	84.44	93.33	92.68	85.71	81	9	90.00	100.00	100.00	90.82
合计	N	6	84	84.44	93.33	92.68	85.71	0	89	90.00	100.00	100.00	90.82
注：a 金标准为按照本研究方法中阳性的判定原则得到的最终结果；P为阳性；N为阴性。

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参考文献(29)

[1]	Zhou MG, Wang HD, Zhu J, et al. Cause - specific mortality for 240 causes in China during 1990 – 2013: a systematic subnational analysis for the global burden of disease study 2013[J]. The Lancet, 2016, 387(10015): 251 – 272. doi: 10.1016/S0140-6736(15)00551-6
[2]	汉聪慧, 曹广进, 张福真, 等. 液相芯片技术与实时荧光定量PCR在检测儿童感染性腹泻病原体的比较[J]. 基础医学与临床, 2020, 40(4): 523 – 527. doi: 10.3969/j.issn.1001-6325.2020.04.018
[3]	Higgins RR, Beniprashad M, Cardona M, et al. Evaluation and verification of the Seeplex Diarrhea-V ACE assay for simultaneous detection of adenovirus, rotavirus, and norovirus genogroups I and II in clinical stool specimens[J]. Journal of Clinical Microbiology, 2011, 49(9): 3154 – 3162. doi: 10.1128/JCM.00599-11
[4]	Damen M, Minnaar R, Glasius P, et al. Real - time PCR with an internal control for detection of all known human adenovirus serotypes[J]. Journal of Clinical Microbiology, 2008, 46(12): 3997 – 4003. doi: 10.1128/JCM.00563-08
[5]	Liu Y, Xu ZQ, Zhang Q, et al. Simultaneous detection of seven enteric viruses associated with acute gastroenteritis by a multiplexed Luminex - based assay[J]. Journal of Clinical Microbiology, 2012, 50(7): 2384 – 2389. doi: 10.1128/JCM.06790-11
[6]	Ishida S, Yoshizumi S, Ikeda T, et al. Sensitive and rapid detection of norovirus using duplex TaqMan reverse transcription - polymerase chain reaction[J]. Journal of Medical Virology, 2008, 80(5): 913 – 920. doi: 10.1002/jmv.21142
[7]	Wang J, Xu ZQ, Niu PH, et al. A two - tube multiplex reverse transcription PCR assay for simultaneous detection of viral and bacterial pathogens of infectious diarrhea[J]. BioMed Research International, 2014, 2014: 648520.
[8]	Jensen AN, Andersen MT, Dalsgaard A, et al. Development of real - time PCR and hybridization methods for detection and identification of thermophilic Campylobacter spp. in pig faecal samples[J]. Journal of Applied Microbiology, 2005, 99(2): 292 – 300. doi: 10.1111/j.1365-2672.2005.02616.x
[9]	Nayak R, Stewart TM, Nawaz MS. PCR identification of Campylobacter coli and Campylobacter jejuni by partial sequencing of virulence genes[J]. Molecular and Cellular Probes, 2005, 19(3): 187 – 193. doi: 10.1016/j.mcp.2004.11.005
[10]	Kubota H, Sakai T, Gawad A, et al. Development of TaqMan - based quantitative PCR for sensitive and selective detection of toxigenic Clostridium difficile in human stools[J]. PLoS One, 2014, 9(10): e111684. doi: 10.1371/journal.pone.0111684
[11]	Liu J, Gratz J, Amour C, et al. A laboratory - developed TaqMan Array Card for simultaneous detection of 19 enteropathogens[J]. Journal of Clinical Microbiology, 2013, 51(2): 472 – 480. doi: 10.1128/JCM.02658-12
[12]	Houser BA, Hattel AL, Jayarao BM. Real - time multiplex polymerase chain reaction assay for rapid detection of Clostridium difficile toxin - encoding strains[J]. Foodborne Pathogens and Disease, 2010, 7(6): 719 – 726. doi: 10.1089/fpd.2009.0483
[13]	Hidaka A, Hokyo T, Arikawa K, et al. Multiplex real - time PCR for exhaustive detection of diarrhoeagenic Escherichia coli[J]. Journal of Applied Microbiology, 2009, 106(2): 410 – 420. doi: 10.1111/j.1365-2672.2008.04043.x
[14]	Villalobo E, Torres A. PCR for detection of Shigella spp. in mayonnaise[J]. Applied and Environmental Microbiology, 1998, 64(4): 1242 – 1245. doi: 10.1128/AEM.64.4.1242-1245.1998
[15]	Kwang J, Littledike ET, Keen JE. Use of the polymerase chain reaction for Salmonella detection[J]. Letters in Applied Microbiology, 1996, 22(1): 46 – 51. doi: 10.1111/j.1472-765X.1996.tb01106.x
[16]	Gugliandolo C, Lentini V, Spanò A, et al. Conventional and molecular methods to detect bacterial pathogens in mussels[J]. Letters in Applied Microbiology, 2011, 52(1): 15 – 21. doi: 10.1111/j.1472-765X.2010.02959.x
[17]	Thoerner P, Bin Kingombe CI, Bögli-Stuber K, et al. PCR detection of virulence genes in Yersinia enterocolitica and Yersinia pseudotuberculosis and investigation of virulence gene distribution[J]. Applied and Environmental Microbiology, 2003, 69(3): 1810 – 1816. doi: 10.1128/AEM.69.3.1810-1816.2003
[18]	Rochelle PA, De Leon R, Stewart MH, et al. Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water[J]. Applied and Environmental Microbiology, 1997, 63(1): 106 – 114. doi: 10.1128/aem.63.1.106-114.1997
[19]	Awad-el-Kariem FM, Warhurst DC, McDonald V. Detection and species identification of Cryptosporidium oocysts using a system based on PCR and endonuclease restriction[J]. Parasitology, 1994, 109(Pt 1): 19 – 22.
[20]	Haque R, Roy S, Siddique A, et al. Multiplex real - time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp[J]. The American Journal of Tropical Medicine and Hygiene, 2007, 76(4): 713 – 717. doi: 10.4269/ajtmh.2007.76.713
[21]	Roy S, Kabir M, Mondal D, et al. Real - time - PCR assay for diagnosis of Entamoeba histolytica infection[J]. Journal of Clinical Microbiology, 2005, 43(5): 2168 – 2172. doi: 10.1128/JCM.43.5.2168-2172.2005
[22]	黎俊宏, 姚萍, 李琼, 等. 多重PCR液相芯片技术在腹泻病原体检测中的应用[J]. 现代预防医学, 2017, 44(18): 3390 – 3394.
[23]	Gosert R, Heininger U, Hirsch HH. Enterovirus detection in patients with acute gastroenteritis in Switzerland[J]. Journal of Medical Virology, 2018, 90(4): 685 – 691. doi: 10.1002/jmv.25005
[24]	Deng JK, Luo X, Wang RL, et al. A comparison of luminex xTAG^® gastrointestinal pathogen panel (xTAG GPP) and routine tests for the detection of enteropathogens circulating in Southern China[J]. Diagnostic Microbiology and Infectious Disease, 2015, 83(3): 325 – 330. doi: 10.1016/j.diagmicrobio.2015.07.024
[25]	Vocale C, Rimoldi SG, Pagani C, et al. Comparative evaluation of the new xTAG GPP multiplex assay in the laboratory diagnosis of acute gastroenteritis. Clinical assessment and potential application from a multicentre Italian study[J]. International Journal of Infectious Diseases, 2015, 34: 33 – 37. doi: 10.1016/j.ijid.2015.02.011
[26]	Huang SH, Lin YF, Tsai MH, et al. Detection of common diarrhea - causing pathogens in Northern Taiwan by multiplex polymerase chain reaction[J]. Medicine, 2018, 97(23): e11006. doi: 10.1097/MD.0000000000011006
[27]	赵新, 兰青阔, 陈锐, 等. 应用微滴数字PCR技术快速检测食用菌中沙门氏菌[J]. 食品与生物技术学报, 2017, 36(3): 315 – 321. doi: 10.3969/j.issn.1673-1689.2017.03.014
[28]	Chhabra P, Gregoricus N, Weinberg GA, et al. Comparison of three multiplex gastrointestinal platforms for the detection of gastroenteritis viruses[J]. Journal of Clinical Virology, 2017, 95: 66 – 71. doi: 10.1016/j.jcv.2017.08.012
[29]	Zhuo R, Cho J, Qiu YY, et al. High genetic variability of norovirus leads to diagnostic test challenges[J]. Journal of Clinical Virology, 2017, 96: 94 – 98. doi: 10.1016/j.jcv.2017.10.003