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Article(id=1250834198978835211, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1250834186500784538, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20260051, pmid=null, cstr=null, oa=null, hot=1, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1768752000000, receivedDateStr=2026-01-19, revisedDate=null, revisedDateStr=null, acceptedDate=1772640000000, acceptedDateStr=2026-03-05, onlineDate=1776151712411, onlineDateStr=2026-04-14, pubDate=1775232000000, pubDateStr=2026-04-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1776151712411, onlineIssueDateStr=2026-04-14, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1776151712411, creator=13701087609, updateTime=1777360085909, updator=13701087609, issue=Issue{id=1250834186500784538, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='4', pageStart='1471', pageEnd='2021', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1776151709437, creator=13701087609, updateTime=1776152261216, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1250836500921922256, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1250834186500784538, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1250836500926116561, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1250834186500784538, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2007, endPage=2021, ext={EN=ArticleExt(id=1250834199909970797, articleId=1250834198978835211, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Development and application of a fluorescence method for Vibrio parahaemolyticus detection based on CRISPR/Cas13a and hybridization chain reaction, columnId=1194702985843413943, journalTitle=Acta Microbiologica Sinica, columnName=Technology and Method, runingTitle=null, highlight=null, articleAbstract=

Objective To develop a fluorescence method for Vibrio parahaemolyticus detection by the combination of CRISPR system and the hybridization chain reaction (HCR), thus achieving rapid, sensitive, and accurate detection of the pathogen. Methods Cascade probe (RP/I) and HCR hairpin structures were first designed according to a specific conserved sequences screened from V. parahaemolyticus. Subsequently, the feasibility, specificity, and sensitivity of the method were evaluated after the optimization of reaction conditions. Furthermore, V. parahaemolyticus-contaminated aquatic products were used to validate the interference resistance of the method. Results The cleavage of CRISPR/Cas13a was activated upon binding to the target RNA (T-RNA), leading to the trans-cleavage of the RP/I cascade probe and the release of I strand. Then, the released I strand subsequently triggered HCR, generating a significant fluorescence signal for target detection. The established method successfully distinguished target sequences with single-base, double-base, and triple-base mismatches and enabled the specific identification of V. parahaemolyticus against other non-target bacteria, including V. alginolyticus, V. vulnificus, V. harveyi, V. cholerae, and Escherichia coli, demonstrating excellent specificity. The assay showed a good linear correlation over a T-RNA concentration range of 25 pmol/L to 10 nmol/L. The corresponding linear regression equation was y=7 236.75×lg CT-RNA-8 590.11 (R2=0.99, C represents the T-RNA concentration and y represents the fluorescence intensity), with the LOD of 1.01 pmol/L. The proposed method enabled rapid detection of RNA extracted from V. parahaemolyticus in various aquatic products, yielding results consistent with those obtained by RT-qPCR. Conclusion The fluorescence method based on CRISPR/Cas13a-HCR established in this study realizes rapid detection of V. parahaemolyticus, demonstrating good sensitivity, specificity, and accuracy.

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E-mail: ZENG Shu,
WAN Yi,
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目的 将CRISPR系统与杂交链式反应(hybridization chain reaction, HCR)结合，构建一种用于副溶血性弧菌快速检测的荧光方法，以实现对病原菌的快速、灵敏、准确检测。 方法 筛选副溶血性弧菌特异性保守序列，设计级联探针(RP/I)及HCR发夹结构，优化反应体系及实验条件，评估该方法的可行性、灵敏度和特异性，并利用副溶血性弧菌污染的水产品验证其抗干扰能力。 结果 仅当靶标存在时，CRISPR/Cas13a才能被激活，反式切割RP/I级联探针，释放出I链并引发HCR反应，产生明显的荧光增强信号。该方法具有良好的检测特异性，可区分单碱基错配、双碱基错配和三碱基错配，并能针对不同菌株有效区分副溶血性弧菌与非靶标菌(如溶藻弧菌、创伤弧菌、哈维氏弧菌、霍乱弧菌和大肠杆菌)。基于纯靶标RNA的检测灵敏度为1.01 pmol/L，在25 pmol/L-10 nmol/L具有良好的线性关系，其回归方程为y=7 236.75×lg CT-RNA-8 590.11，R2=0.99。本研究方法可快速检测多种水产品中副溶血性弧菌提取的RNA，检测结果与实时荧光定量逆转录PCR (real-time reverse transcription quantitative PCR, RT-qPCR)一致。 结论 本研究建立的CRISPR/Cas13a-HCR荧光检测方法可快速、准确地检测副溶血性弧菌，且具有良好的灵敏度、特异性和准确性。

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作者贡献声明

黄梦琴：实验操作、数据处理和论文撰写；辛煜：数据分析、验证；符丹凤：样本获取与处理；刘子扬：数据呈现；彭道云：文献调研；曾姝：方法论、论文审阅和修改、基金获取；万逸：方法论、论文审阅和修改；崔倩：细菌培养；陈菲：文献调研、细菌培养。

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articleId=1250834198978835211, language=EN, label=Figure 2, caption=The feasibility of CRISPR/Cas13a-HCR assay for the detection of T-RNA. A: Feasibility characterization of the Cas13a cleavage reaction using 12% Native-PAGE; B: Feasibility assessment of HCR reaction initiated by isolated I-chain using 12% Native-PAGE; C: Characterization of the CRISPR/Cas13a-HCR-based reaction using 12% Native-PAGE; D: Fluorescence emission spectra of the CRISPR/Cas13a-HCR-based reaction., figureFileSmall=6KtwPx8DSIl7AqFTgUM99w==, figureFileBig=Xl90jzF4JCevMc/2h98tJg==, tableContent=null), ArticleFig(id=1250879401655742886, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=CN, label=图2, caption=CRISPR/Cas13a-HCR检测T-RNA可行性分析, figureFileSmall=6KtwPx8DSIl7AqFTgUM99w==, figureFileBig=Xl90jzF4JCevMc/2h98tJg==, tableContent=null), ArticleFig(id=1250879401819320746, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=EN, label=Figure 3, caption=Optimization of experimental conditions for the CRISPR/Cas13a-HCR assay. A: RP/I ratio; B: RP/I concentration; C: Cas13a concentration; D: crRNA concentration; E: Incubation time for CRISPR/Cas13a assay; F: Incubation time for HCR assay. The concentration of T-RNA was 50 nmol/L., figureFileSmall=aA+6EMrezWestrzz8JMrhg==, figureFileBig=V6GYZpWDE5F4IRt9z8MUkg==, tableContent=null), ArticleFig(id=1250879401940955568, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=CN, label=图3, caption=CRISPR/Cas13a-HCR的条件优化, figureFileSmall=aA+6EMrezWestrzz8JMrhg==, figureFileBig=V6GYZpWDE5F4IRt9z8MUkg==, tableContent=null), ArticleFig(id=1250879402083561908, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=EN, label=Figure 4, caption=Performance of CRISPR/Cas13a-HCR assay for T-RNA detection. A: Fluorescence emission spectra of different T-RNA concentrations from 0 to 10 nmol/L; B: Linear analysis for T-RNA detection., figureFileSmall=mWwiu61RGE6P+TMtX71eYA==, figureFileBig=4PkXoYQCXPRa2xqvXklt/w==, tableContent=null), ArticleFig(id=1250879402196808121, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=CN, label=图4, caption=CRISPR/Cas13a-HCR的灵敏度分析, figureFileSmall=mWwiu61RGE6P+TMtX71eYA==, figureFileBig=4PkXoYQCXPRa2xqvXklt/w==, tableContent=null), ArticleFig(id=1250879402314248637, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=EN, label=Figure 5, caption=Specificity analysis of the CRISPR/Cas13a-HCR assay. A: T-RNA and its mismatched base sequences; B: Fluorescence emission spectra of the mismatched sequences; C: Significance analysis of the mismatched sequences; D: Total RNA from target and non-target bacteria; E: Fluorescence emission spectra of the total bacterial RNA; F: Significance analysis of the total bacterial RNA. **** indicates a significant difference compared to the control group (P<0.000 1); ns indicates no statistical difference (P>0.05); All data are from three independent experiments (n=3)., figureFileSmall=s71GhCc5s1o+JW/IQN34Eg==, figureFileBig=H7o4dd1zA+LkS+ranDO+fA==, tableContent=null), ArticleFig(id=1250879402452660676, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=CN, label=图5, caption=CRISPR/Cas13a-HCR实验的特异性分析, figureFileSmall=s71GhCc5s1o+JW/IQN34Eg==, figureFileBig=H7o4dd1zA+LkS+ranDO+fA==, tableContent=null), ArticleFig(id=1250879402561712586, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=EN, label=Figure 6, caption=Analysis of complex samples. A: Schematic diagram of the sample processing procedure for complex samples; B: Significance analysis based on the CRISPR/Cas13a-HCR strategy; C: Significance analysis based on the RT-qPCR strategy (Ct values for RT-qPCR-negative groups were undetectable and were set to 40). **** indicates a significant difference compared to the negative group (P<0.000 1); All data are from three independent experiments (n=3)., figureFileSmall=JFLiTbBTCKARJByVHMFDkw==, figureFileBig=hLCmUHPcTjc/LhE1FrC4Gg==, tableContent=null), ArticleFig(id=1250879402679153105, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=CN, label=图6, caption=复杂样本分析, figureFileSmall=JFLiTbBTCKARJByVHMFDkw==, figureFileBig=hLCmUHPcTjc/LhE1FrC4Gg==, tableContent=null), ArticleFig(id=1250879402763039189, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=EN, label=Table 1, caption=

Strain information

, figureFileSmall=null, figureFileBig=null, tableContent=
Strain nameStrain collection number
副溶血性弧菌Vibrio parahaemolyticusATCC 17802
溶藻弧菌Vibrio alginolyticusATCC 33787
创伤弧菌Vibrio vulnificusATCC 27562
哈维氏弧菌Vibrio harveyiATCC BAA-1117
霍乱弧菌Vibrio choleraeCICC 23794
大肠杆菌Escherichia coliCICC 10899
), ArticleFig(id=1250879402876285404, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=CN, label=表1, caption=

菌株信息

, figureFileSmall=null, figureFileBig=null, tableContent=
Strain nameStrain collection number
副溶血性弧菌Vibrio parahaemolyticusATCC 17802
溶藻弧菌Vibrio alginolyticusATCC 33787
创伤弧菌Vibrio vulnificusATCC 27562
哈维氏弧菌Vibrio harveyiATCC BAA-1117
霍乱弧菌Vibrio choleraeCICC 23794
大肠杆菌Escherichia coliCICC 10899
), ArticleFig(id=1250879402943394272, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=EN, label=Table 2, caption=

Nucleotide sequence used in this study

, figureFileSmall=null, figureFileBig=null, tableContent=
Nucleotide nameNucleotide sequences (5′→3′)
T-RNAUGAACCAGAAGCGCCAGUAGUACCUGAA
crRNAGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUUCAGGUACUACUGGCGCUUCUGGUUCA
RPUUAACCCACGCUUUUUCAUCCUAGACU
IAGTCTAGGATGTCGCGTGGGTTAA
H1.1TTAACCCACGCGACATCCTAGACTCAAAGTAGTCTAGGATGTCGCGTG
H1TTAACCCACGCGACA/i6FAMdT/CCTAGACTCAAAGTAGTCTAGGA/iBHQ1dT/GTCGCGTG
H2AGTCTAGGATGTCGCGTGGGTTAACACGCGACATCCTAGACTACTTTG
RT-qPCR F1GTCTTTAGCGACGACTTC
RT-qPCR R1GTAAACAGCAGTACGCAA
), ArticleFig(id=1250879403027280356, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1250834198978835211, language=CN, label=表2, caption=

本研究所用核酸序列

, figureFileSmall=null, figureFileBig=null, tableContent=
Nucleotide nameNucleotide sequences (5′→3′)
T-RNAUGAACCAGAAGCGCCAGUAGUACCUGAA
crRNAGAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUUCAGGUACUACUGGCGCUUCUGGUUCA
RPUUAACCCACGCUUUUUCAUCCUAGACU
IAGTCTAGGATGTCGCGTGGGTTAA
H1.1TTAACCCACGCGACATCCTAGACTCAAAGTAGTCTAGGATGTCGCGTG
H1TTAACCCACGCGACA/i6FAMdT/CCTAGACTCAAAGTAGTCTAGGA/iBHQ1dT/GTCGCGTG
H2AGTCTAGGATGTCGCGTGGGTTAACACGCGACATCCTAGACTACTTTG
RT-qPCR F1GTCTTTAGCGACGACTTC
RT-qPCR R1GTAAACAGCAGTACGCAA
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基于CRISPR/Cas13a-HCR的副溶血性弧菌荧光检测方法构建及应用
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黄梦琴 1 , 辛煜 1 , 符丹凤 2 , 刘子扬 1 , 彭道云 3 , 曾姝 4 , 万逸 4 , 崔倩 5 , 陈菲 6
微生物学报 | 技术与方法 2026,66(4): 2007-2021
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微生物学报 | 技术与方法 2026, 66(4): 2007-2021
基于CRISPR/Cas13a-HCR的副溶血性弧菌荧光检测方法构建及应用
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黄梦琴1, 辛煜1, 符丹凤2, 刘子扬1, 彭道云3, 曾姝4 , 万逸4 , 崔倩5, 陈菲6
作者信息
  • 1.海南大学 海洋生物与水产学院，海南 海口
  • 2.海南省水产品质量安全检测中心(海南省水产技术推广站)，海南 海口
  • 3.海南大学 生物医学工程学院，海南 海口
  • 4.海南大学 海洋科学学院(南海海洋资源利用国家重点实验室)，海南 海口
  • 5.海南大学 生命健康学院，海南 海口
  • 6.海南大学 食品科学与工程学院，海南 海口
Development and application of a fluorescence method for Vibrio parahaemolyticus detection based on CRISPR/Cas13a and hybridization chain reaction
Mengqin HUANG1, Yu XIN1, Danfeng FU2, Ziyang LIU1, Daoyun PENG3, Shu ZENG4 , Yi WAN4 , Qian CUI5, Fei CHEN6
Affiliations
  • 1.School of Marine Biology and Fisheries, Hainan University, Haikou, Hainan, China
  • 2.Hainan Provincial Aquatic Product Quality and Safety Inspection Center (Hainan Provincial Fisheries Technology Extension Station), Haikou, Hainan, China
  • 3.School of Biomedical Engineering, Hainan University, Haikou, Hainan, China
  • 4.School of Marine Sciences (State Key Laboratory of Marine Resource Utilization in South China Sea), Hainan University, Haikou, Hainan, China
  • 5.School of Life and Health Sciences, Hainan University, Haikou, Hainan, China
  • 6.School of Food Science and Engineering, Hainan University, Haikou, Hainan, China
出版时间: 2026-04-04 doi: 10.13343/j.cnki.wsxb.20260051
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摘要
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目的 将CRISPR系统与杂交链式反应(hybridization chain reaction, HCR)结合，构建一种用于副溶血性弧菌快速检测的荧光方法，以实现对病原菌的快速、灵敏、准确检测。 方法 筛选副溶血性弧菌特异性保守序列，设计级联探针(RP/I)及HCR发夹结构，优化反应体系及实验条件，评估该方法的可行性、灵敏度和特异性，并利用副溶血性弧菌污染的水产品验证其抗干扰能力。 结果 仅当靶标存在时，CRISPR/Cas13a才能被激活，反式切割RP/I级联探针，释放出I链并引发HCR反应，产生明显的荧光增强信号。该方法具有良好的检测特异性，可区分单碱基错配、双碱基错配和三碱基错配，并能针对不同菌株有效区分副溶血性弧菌与非靶标菌(如溶藻弧菌、创伤弧菌、哈维氏弧菌、霍乱弧菌和大肠杆菌)。基于纯靶标RNA的检测灵敏度为1.01 pmol/L，在25 pmol/L-10 nmol/L具有良好的线性关系，其回归方程为y=7 236.75×lg CT-RNA-8 590.11，R2=0.99。本研究方法可快速检测多种水产品中副溶血性弧菌提取的RNA，检测结果与实时荧光定量逆转录PCR (real-time reverse transcription quantitative PCR, RT-qPCR)一致。 结论 本研究建立的CRISPR/Cas13a-HCR荧光检测方法可快速、准确地检测副溶血性弧菌，且具有良好的灵敏度、特异性和准确性。

副溶血性弧菌  /  CRISPR/Cas13a  /  杂交链式反应  /  荧光检测

Objective To develop a fluorescence method for Vibrio parahaemolyticus detection by the combination of CRISPR system and the hybridization chain reaction (HCR), thus achieving rapid, sensitive, and accurate detection of the pathogen. Methods Cascade probe (RP/I) and HCR hairpin structures were first designed according to a specific conserved sequences screened from V. parahaemolyticus. Subsequently, the feasibility, specificity, and sensitivity of the method were evaluated after the optimization of reaction conditions. Furthermore, V. parahaemolyticus-contaminated aquatic products were used to validate the interference resistance of the method. Results The cleavage of CRISPR/Cas13a was activated upon binding to the target RNA (T-RNA), leading to the trans-cleavage of the RP/I cascade probe and the release of I strand. Then, the released I strand subsequently triggered HCR, generating a significant fluorescence signal for target detection. The established method successfully distinguished target sequences with single-base, double-base, and triple-base mismatches and enabled the specific identification of V. parahaemolyticus against other non-target bacteria, including V. alginolyticus, V. vulnificus, V. harveyi, V. cholerae, and Escherichia coli, demonstrating excellent specificity. The assay showed a good linear correlation over a T-RNA concentration range of 25 pmol/L to 10 nmol/L. The corresponding linear regression equation was y=7 236.75×lg CT-RNA-8 590.11 (R2=0.99, C represents the T-RNA concentration and y represents the fluorescence intensity), with the LOD of 1.01 pmol/L. The proposed method enabled rapid detection of RNA extracted from V. parahaemolyticus in various aquatic products, yielding results consistent with those obtained by RT-qPCR. Conclusion The fluorescence method based on CRISPR/Cas13a-HCR established in this study realizes rapid detection of V. parahaemolyticus, demonstrating good sensitivity, specificity, and accuracy.

Vibrio parahaemolyticus  /  CRISPR/Cas13a  /  hybridization chain reaction  /  fuorescence detection
黄梦琴, 辛煜, 符丹凤, 刘子扬, 彭道云, 曾姝, 万逸, 崔倩, 陈菲. 基于CRISPR/Cas13a-HCR的副溶血性弧菌荧光检测方法构建及应用. 微生物学报, 2026 , 66 (4) : 2007 -2021 . DOI: 10.13343/j.cnki.wsxb.20260051
Mengqin HUANG, Yu XIN, Danfeng FU, Ziyang LIU, Daoyun PENG, Shu ZENG, Yi WAN, Qian CUI, Fei CHEN. Development and application of a fluorescence method for Vibrio parahaemolyticus detection based on CRISPR/Cas13a and hybridization chain reaction[J]. Acta Microbiologica Sinica, 2026 , 66 (4) : 2007 -2021 . DOI: 10.13343/j.cnki.wsxb.20260051
正文
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食源性疾病是全球范围内一项重大的公共卫生挑战。细菌性病原体引发的感染在疾病负担中占据重要地位，是导致腹泻、败血症等严重临床后果的主要因素之一[1]。副溶血性弧菌属于弧菌科、弧菌属，是一种革兰氏阴性菌，同时也是引发生物性食源性疾病的最主要致病菌之一，作为全球沿海水域最为普遍的细菌性病原体，它对人类健康和渔业构成极大威胁[2-3]。其致病因子包括热不稳定溶血素(thermolabile hemolysin, Tlh)基因、热稳定直接溶血素(thermostable direct hemolysin, Tdh)以及与Tdh相关溶血素(tdh-related hemolysin, Trh)基因的产物，还有与β溶血相关的因子等，这些因子会直接或间接引发人体伤口感染、原发性败血症和胃肠炎[4]。因此，亟需开发灵敏、高效的副溶血性弧菌检测技术。
近年来，CRISPR/Cas系统因能特异性识别并切割核酸靶标，已成为基因编辑和微生物诊断领域的研究热点。CRISPR/Cas系统由簇状规则间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)和CRISPR相关蛋白(CRISPR-associated proteins, Cas)构成[5-6]。其中，CRISPR/Cas13a (前称C2c2)是一种RNA靶向的核酸酶，其crRNA由直接重复序列和间隔序列组成，间隔区序列可通过编程与靶标互补，识别靶标RNA后，激活的Cas13a可非特异性切割周围的单链RNA (single-stranded RNA, ssRNA) (也称为旁支切割)，从而提供特定的RNA检测平台[7-10]。杂交链式反应(hybridization chain reaction, HCR)是一种无酶的核酸聚合反应，作为一种无酶、等温、可编程的核酸自组装放大技术，仅需简单的温控设备即可完成反应。该技术无需辅助酶参与，可大大降低实验成本，利用HCR的信号转导和无酶扩增能力能够实现靶分子的信号放大[11-12]。
本研究以副溶血性弧菌特异性toxR基因为靶点，结合CRISPR/Cas13a系统和HCR技术进行级联信号转导放大，以期构建副溶血性弧菌检测新平台。
1 材料与方法
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1.1 菌株
本课题组共购买6株实验菌株，分别为副溶血性弧菌、溶藻弧菌、创伤弧菌、哈维氏弧菌、霍乱弧菌以及大肠杆菌，菌株信息如表1所示。
1.2 靶标RNA、crRNA、级联探针和发夹探针的设计
从美国国家生物技术信息中心(National Center for Biotechnology Information, NCBI)数据库(https://www.ncbi.nlm.nih.gov/)获取toxR基因序列，使用MEGA-X软件将副溶血性弧菌与其他弧菌进行序列比对，选取相对保守的区域作为检测靶区，使用NCBI BLAST验证靶标RNA (target RNA, T-RNA)的特异性。crRNA序列由直接重复区和间隔区组成，设计间隔区序列与T-RNA序列互补。T-RNA、crRNA、RP、I、H1.1、H1和H2链由生工生物工程(上海)股份有限公司合成，核酸序列见表2。
1.3 RP/I探针制备
为形成RNA与DNA的杂交体RP/I级联探针，将终浓度为2.5 μmol/L的I链(DNA)和2.8 μmol/L的RP链(RNA)、1×Annealing buffer用无酶水配至所需体积，混匀后于95 ℃退火5 min，随后缓慢降温至37 ℃孵育1.5 h，最后置于8 ℃保存。
1.4 Cas13a切割反应
Cas13a切割反应体系(20 μL)：T-RNA 3 μL，Cas13a (400 nmol/L) 3 μL，crRNA (200 nmol/L) 3 μL，RP/I探针(2.5 μmol/L) 3 μL，5×Cas13a反应缓冲液4 μL，DEPC水4 μL。在37 ℃孵育30 min后用于后续实验。
1.5 HCR反应及荧光测定
HCR反应体系(30 μL)：在1.4节反应体系中加入10 μL H1和H2反应液，使H1和H2终浓度均为400 nmol/L (即4 μmol/L)，加入1×HCR反应缓冲液(20 mmol/L Tris-HCl, 5 mmol/L MgCl2, 300 mmol/L NaCl, pH 7.60)。将反应体系于37 ℃下反应2 h，使用酶标仪(BioTek公司)记录荧光。H1、H2在反应前进行预处理：95 ℃退火5 min，立即于4 ℃保存备用。
1.6 聚丙烯酰胺凝胶电泳分析
使用非变性聚丙烯酰胺凝胶电泳(native polyacrylamide gel electrophoresis, Native-PAGE)验证RP/I探针、Cas13a切割产物及HCR扩增产物。电压设置为110 V，缓冲液为1×TBE。电泳结束后，小心取出凝胶，置于1×Super Red染色液中避光染色10 min，然后使用凝胶成像系统在紫外下进行成像、拍照并记录数据结果。
1.7 RNA提取
将50 μL甘油保存的副溶血性弧菌接种至5 mL 3%氯化钠碱性蛋白胨水(3% NaCl alkaline peptone water, 3% NaCl APW)；将50 μL甘油-80 ℃保存的溶藻弧菌、创伤弧菌、哈维氏弧菌、霍乱弧菌和大肠杆菌分别加入1%氯化钠碱性蛋白胨水(1% NaCl alkaline peptone water, 1% NaCl APW)中，在37 ℃、180 r/min条件下培养12 h，分别取3 mL用于RNA提取，使用细菌RNA提取试剂盒(Omega公司)按说明书操作提取RNA，使用NanoDrop超微量紫外分光光度计(ThermoFisher Scientific公司)测定RNA浓度，1%琼脂糖凝胶电泳分析RNA提取产物质量，提取的RNA置于-80 ℃保存。
1.8 副溶血性弧菌添加样本制备
南美白对虾(Litopenaeus vannamei)、珍珠龙胆石斑鱼(Epinephelus fuscoguttatus ♀×Epinephelus lanceolatus ♂)、太平洋牡蛎(Crassostrea gigas)，购自海南本地生鲜超市。解剖分离对虾的肝胰腺组织、石斑鱼的肠组织、牡蛎的内脏团组织，分别称取2.5 g组织研磨后置于50 mL无菌离心管中，加入22.5 mL 3%氯化钠碱性蛋白胨水(3% NaCl alkaline peptone water, 3% NaCl APW)，均质处理2 min。将121 ℃灭菌15 min灭菌后的组织样本进行分组处理，阳性组中加入2.5 mL 108 CFU/mL的副溶血性弧菌菌液，使终浓度达到107 CFU/mL，而阴性组中加入2.5 mL灭菌的0.9%生理盐水。将制备的阳性组和阴性组组织样本旋涡振荡混匀后，取3 mL进行组织样本RNA提取，提取的RNA置于-80 ℃保存。
1.9 统计分析
实验独立重复3次(n=3)，数据以平均值±标准差(SD)表示。采用Origin 2021、GraphPad Prism 9.5软件进行数据分析，单因素多组间比较采用单因素ANOVA方差分析(one-way ANOVA)，多重比较采用Dunnett’s多重比较方法。P<0.05认为差异具有统计学意义(ns表示P>0.05；****表示P<0.000 1)。
2 结果与分析
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2.1 检测原理
通过序列比对，选择副溶血弧菌特异性保守序列T-RNA (28 nt)作为靶标序列，结合Cas13a设计crRNA。在有靶标存在的情况下，T-RNA与crRNA结合，激活Cas13a活性，反式切割RP1/I级联探针上的U碱基，从而释放I链，引发HCR反应。该反应依次循环打开H1和H2的发夹结构，形成带有缺口的长链双链HCR产物。H1上标记的FAM基团在发夹结构关闭时，因与BHQ1基团邻近而被淬灭；打开后，随着BHQ1基团的远离，FAM的荧光会再次恢复。随着HCR反应的进行，打开的H1越多，FAM基团累计恢复的荧光信号越强，从而实现靶标菌的荧光检测，检测原理示意图见图1。
2.2 可行性分析
通过12% Native-PAGE验证Cas13a切割反应体系中靶标T-RNA能否结合crRNA，激活Cas13a切割RP/I探针。如图2A所示，泳道3和泳道4分别为RP单链和I单链，泳道5为RP和I链的退火产物，其电泳速度明显慢于泳道3和4，说明形成了RP/I探针。加入T-RNA、crRNA和Cas13a蛋白后泳道6中RP/I探针条带消失，并出现了一条属于I链的条带；与泳道6对比，泳道7中RP/I探针条带未消失，这说明虽然添加了crRNA和Cas13a蛋白，但无T-RNA时Cas13a的反式切割活性未能激活，无法切割RP/I探针上的U碱基。这些数据表明，T-RNA可结合crRNA激活Cas13a活性，反式切割RP/I探针从而释放I链。
本研究通过12% Native-PAGE验证单独I链能否引发HCR反应，结果如图2B所示。泳道1、2、4分别为单独的发夹H1.1、发夹H2及引发链I，均仅呈现单一的条带，对应各自的分子量大小。泳道3为H1.1与H2的混合物，也仅观察到单一条带，且迁移速率与单独的H1.1和H2基本一致，表明在无引发链存在的情况下，H1.1与H2之间不会发生非特异性组装或自发开环反应，背景信号较低。泳道5为H1.1、H2与I链的混合物，结果显示出现多条分子量大小不一的高分子量新条带。这是由于I链首先与发夹H1.1的部分互补序列结合，打开H1.1的发夹结构，暴露其黏性末端；该黏性末端进而与发夹H2的部分区域互补，打开H2的发夹结构；H2暴露的黏性末端又可继续与下一个游离的H1.1发夹结合，如此交替循环，形成具有重复单元的长链双链DNA产物，即HCR扩增产物。上述结果表明，单独I链可有效触发HCR级联反应，产生多条高分子量的长链双链DNA产物。
接下来进一步验证切割释放的I链能否引发后续级联的HCR反应。如图2C所示，泳道1和泳道2分别为H1.1单链和H2单链，泳道7为H1.1和H2的混合物，仅有一条对应H1.1和H2混合物的条带，说明H1.1和H2在无引发剂时相对稳定，不会自发组装产生背景条带；即使添加了RP/I探针(泳道8)，也基本未产生杂带。添加T-RNA和对应的crRNA及蛋白后(泳道9)，泳道口以及附近出现分子量大小不一的产物带，表明切割释放的I链引发了后续HCR级联反应。图2D荧光光谱数据进一步证实了这一点，仅有T-RNA存在时，体系在518 nm处的荧光显著高于无T-RNA组以及其他对照组，且其在518 nm处的荧光值是无T-RNA对照组的58.76倍。这些结果说明本方法具有用于副溶血弧菌检测的潜力。
2.3 条件优化
为了实现更好的性能，对CRISPR/Cas13a-HCR的实验条件进行了优化。首先优化了关键组分RP/I探针中RP与I的杂交比例。RP与I杂交比例分别为1.0:1、1.2:1、1.4:1、1.6:1，随着杂交比例的增加，518 nm处T-RNA组与对照组的荧光比值先升高后降低，其对照组背景信号的荧光值逐渐降低，在1.2:1时，518 nm处T-RNA组与对照组的荧光比值达到最大，因此选择RP与I最佳杂交比例为1.2:1 (图3A)。随后优化了RP/I探针在切割体系中的反应浓度，随着探针浓度从225 nmol/L增至450 nmol/L，518 nm处T-RNA组与对照组的荧光值比值先增加后下降，在375 nmol/L时达到最大(图3B)，因此选择该浓度用于后续实验。
接着，优化了Cas13a的反应浓度，将Cas13a浓度从45.0 nmol/L增至75.0 nmol/L，T-RNA组与对照组的荧光值比值先增加后下降最后稳定(图3C)，最后选择60.0 nmol/L作为Cas13a的最佳反应浓度。随着crRNA浓度在15.0-45.0 nmol/L范围内增加，T-RNA组与对照组的荧光比值先增加，后在30.0 nmol/L时达到稳定状态(图3D)，因此选择30.0 nmol/L作为crRNA的最佳反应浓度。随后对Cas13a反应时间和HCR反应时间进行了优化。随着Cas13a反应时间延长，荧光比值增加后在40 min处稳定(图3E)。对HCR反应时间进行2 h的荧光动力学检测，其荧光信号随时间逐渐增强，并在40 min时达到稳定(图3F)。因此，Cas13a反应时间和HCR反应最佳反应时间均为40 min。
2.4 灵敏度
在上述最佳条件下，基于CRISPR/Cas13a和HCR级联策略对不同浓度的T-RNA进行灵敏度评估。如图4A所示，在0-10 nmol/L浓度范围内，随着靶标浓度升高，荧光信号逐渐增强。然而，在靶标浓度范围为25 pmol/L-10 nmol/L时，荧光强度与T-RNA浓度的对数呈线性相关如图4B所示。其线性关系为y=7 236.75×lg CT-RNA-8 590.11，R2=0.99，基于3σ/k准则计算出检测限(limit of detection, LOD)为1.01 pmol/L，其中σ为7个空白样本的标准偏差，k为标准曲线的斜率，表明CRISPR/Cas13a-HCR具有良好的检测灵敏度。
2.5 特异性
为验证CRISPR/Cas13a-HCR策略检测的特异性，根据T-RNA分别设计了单碱基(M1)、双碱基(M2)和三碱基(M3)错配链，错配位置见图5A。结果如图5B、5C所示，添加1 nmol/L T-RNA后，荧光信号显著增强(****: P<0.000 1)，而单碱基、双碱基和三碱基错配链未引起显著的荧光变化(ns: P>0.05)，表明Cas13a-HCR具有单碱基识别能力。另外，如图5D所示，针对5种弧菌属弧菌(副溶血性弧菌、溶藻弧菌、创伤弧菌、哈维氏弧菌和霍乱弧菌)以及大肠杆菌提取总RNA，以进一步评估该策略的特异性检测能力，荧光结果如图5E、5F所示，仅副溶血性弧菌组荧光信号显著增强(****: P<0.000 1)，其他5组荧光无显著增强(ns: P>0.05)，说明CRISPR/Cas13a-HCR能区分副溶血性弧菌及近缘弧菌或其他细菌，进一步证明该检测方法的特异性良好。
2.6 实际样本
为评估CRISPR/Cas13a-HCR方法在复杂样本中的检测能力，对27份组织样本(南美白对虾的肝胰腺组织、珍珠龙胆石斑鱼的肠组织以及太平洋牡蛎的内脏团组织)进行检测。复杂样本处理过程示意图见图6A。CRISPR/Cas13a-HCR检测结果如图6B所示，阳性样本组的荧光信号强度显著高于阴性样本组的荧光信号强度，表明CRISPR/Cas13a-HCR方法可显著区分所有阳性样本与阴性样本。RT-qPCR复杂样本分析结果如图6C所示，所有阳性样本组产生的Ct值均显著低于40，而所有阴性样本组均无Ct值(阴性样本组Ct值设为RT-qPCR程序循环次数40)，CRISPR/Cas13a-HCR与RT-qPCR检测结果一致。上述结果表明CRISPR/Cas13a-HCR对复杂样本的检测具有良好的准确率。
3 讨论与结论
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据联合国粮农组织(Food and Agriculture Organization of the United Nations, FAO)发布的《2024年世界渔业和水产养殖状况》报告显示，2022年全球渔业和水产养殖总产量为2.232亿t，其中水生动物产量为1.854亿t，藻类产量为3 780万t，全球水产养殖产量为1.309亿t，全球渔业水产养殖动物类产量首次超过捕捞渔业动物类产量，其中中国的水生动物产量占全球总量的36%，目前中国已成为全球最大的水产养殖生产国[13]。然而，随着集约化养殖的发展，养殖水域环境恶化，不同季节、不同养殖水域的水产病害频发，水产病害问题已成为制约我国渔业发展的重要因素，弧菌病原中副溶血性弧菌是影响虾蟹类细菌性疾病的主要病原，如对虾的急性肝胰腺坏死病、幼体弧菌病以及红腿病[14-17]。开发灵敏、高效的病原检测技术对我国渔业的可持续发展具有重要的现实意义。
目前，基于RNA的病原检测技术一般需要逆转录过程，如实时荧光定量逆转录PCR (real-time reverse transcription quantitative PCR, RT-qPCR)，首先需要设计多对引物并验证其特异性，以避免假阳性结果。其次，检测环境需洁净，否则靶标RNA降解可能导致模板不完整，从而产生假阴性结果；或阴性样本污染可能引发假阳性结果，这2种结果在预实验中均出现过。另外，PCR需要复杂的温度循环，对实验仪器要求严格[18]。与RT-qPCR相比，本研究同样涉及菌总RNA的提取与-80 ℃保存，但省去了引物设计与逆转录步骤，简化了实验复杂程度。本研究直接将易降解的RNA信号转导为DNA信号，且靶标长度仅需28 nt，反应过程为等温反应(37 ℃)，这在一定程度上提升了信号输出的稳定性及准确性。本研究方法可行的原因主要有以下2点。(1) CRISPR/Cas13a系统具有高度特异性。依赖向导RNA (CRISPR-associated RNA, crRNA)与靶标RNA完全互补后，激活Cas13a蛋白的反式切割活性，从而非特异性切割周围单链RNA (single-stranded RNA, ssRNA)[19-21]。本研究构建了一种RNA与DNA杂交的级联探针(RP/I)，被靶标RNA (T-RNA)激活的CRISPR/Cas13a能切割RP/I探针并成功释放出引发链I (DNA)。(2) 为了进一步实现信号放大输出，本研究引入了HCR技术，通过一对DNA发夹探针(H1和H2)在引发链I的作用下发生级联杂交，形成长的双链DNA，其中H1标记荧光FAM基团，因此能输出强大的荧光信号(图2B、2D)。
Cas13a蛋白在CRISPR RNA (crRNA)的引导下，能够高度特异性地识别并结合目标RNA序列。这种识别机制的优势在于严格遵守碱基互补配对原则，使其能够精确区分单碱基突变，为检测提供了极高的准确性。HCR反应是一种无酶等温扩增技术，操作简单、成本低廉。HCR反应能将单个目标结合事件转换为长链DNA聚合物的形成，每个聚合物上可以负载大量的信号分子(如荧光基团、拉曼探针)，实现信号的级联放大。Cas13a提供了良好的特异性，而HCR则提供了灵活的信号放大能力。将两者级联，利用Cas13a的特异性识别切割来产生触发器，进而启动HCR的二次信号放大，从而实现超灵敏、高特异性检测[22-26]。本研究的核心创新点在于利用CRISPR/Cas13a的特异性识别和HCR的级联放大机制，将单个RNA识别转换为DNA信号，将检测限(LOD)降至1.01 pmol/L (图4)。该性能与其他研究方法相似，例如，Lysne等[27]开发的量子点(quantum dot, QD)分子信标(quantum dot-molecular beacon, QD-MB)平台集成Förster共振能量转移(Förster resonance energy transfer, FRET)与CRISPR-Cas13a、Bruch团队[28]开发的CRISPR/Cas13a驱动的电化学微流控生物传感器以及Zhou等[8]开发的CRISPR/Cas13a与HCR可视化检测平台，其检测限LOD结果都在pmol/L水平。也有将CRISPR/Cas13a与催化发夹组装(CHA)技术结合的电化学生物传感平台，在无靶标预扩增下其灵敏度可达fmol/L水平[29]。
尽管本研究成功建立了快速、灵敏、特异性的CRISPR/Cas13a-HCR检测方法，但仍存在一定局限性：(1) 未实现一锅法操作，CRISPR/Cas13a-HCR的两步法增加了开盖污染的风险，步骤相对繁琐；(2) 与集成核酸扩增技术的CRISPR/Cas13a系统相比，灵敏度性能还有提升空间，如基于CRISPR/Cas13a系统的SHERLOCK[30]以及超分支滚动环扩增(hyper-branched rolling circle amplification, HRCA)[31]，其检测灵敏度甚至可达amol/L级别；(3) 该检测平台针对的是单一靶标，而水产病害常由多种病原共同引起，当前尚未满足水产病害多靶标检测需求。针对上述局限性，未来可从以下3个方面进行优化。(1) 引入DNA四面体纳米结构提升灵敏度性能[32-34]。本研究的HCR体系依赖于自由扩散的发夹探针之间的随机碰撞，这导致反应效率较低、耗时较长，限制了其在高通量快速检测中的应用。然而，即使设计再精密的发夹结构，也可能因发夹的非特异性打开而产生背景信号，特别是在复杂的生物基质中，这可能会降低检测的信噪比。若能将DNA四面体设计为发夹H1、H2的信号载体，利用DNA四面体的4个顶点连接发夹H1、H2，利用DNA四面体的刚性结构和空间限域效应可增加局部分子反应浓度，有效提升信号强度。(2) 设计多重crRNA与多元HCR系统，利用不同荧光基团进行标记，实现多靶标检测以提升检测效率。(3) 开发微流控芯片生物传感平台，通过设计加样室、Cas反应室和HCR反应室的微流控芯片，实现两步反应的时空隔离，开发全集成、一体化、多通道的便携式信号检测装置，进一步降低操作门槛和使用成本[35-36]。
综上所述，本研究聚焦病原微生物诊断的高灵敏、快速检测需求，成功构建了一种CRISPR/Cas13a-HCR诊断方法。CRISPR/Cas13a-HCR方法无需靶标预扩增、引物设计和复杂的温度循环程序即可实现1.01 pmol/L的RNA检测灵敏度，反应温度为37 ℃，检测时间约2 h。CRISPR/Cas13a-HCR不仅能有效区分副溶血性弧菌与近缘弧菌或其他细菌，更具有单碱基识别能力，表现出良好的特异性。该方法在3种模拟的复杂样本(27份)中均达到100%准确率，为微生物诊断提供了新的技术选择。
基金
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  • 国家自然科学基金(22204036)
  • 海南省科技专项资助(ZDYF2025XDNY066)
文献
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2026年第66卷第4期
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doi: 10.13343/j.cnki.wsxb.20260051
  • 接收时间：2026-01-19
  • 首发时间：2026-04-14
  • 出版时间：2026-04-04
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  • 收稿日期：2026-01-19
  • 录用日期：2026-03-05
基金
National Natural Science Foundation of China(22204036)
国家自然科学基金(22204036)
Hainan Provincial Science and Technology Special Fund(ZDYF2025XDNY066)
海南省科技专项资助(ZDYF2025XDNY066)
作者信息
    1.海南大学 海洋生物与水产学院，海南 海口
    2.海南省水产品质量安全检测中心(海南省水产技术推广站)，海南 海口
    3.海南大学 生物医学工程学院，海南 海口
    4.海南大学 海洋科学学院(南海海洋资源利用国家重点实验室)，海南 海口
    5.海南大学 生命健康学院，海南 海口
    6.海南大学 食品科学与工程学院，海南 海口
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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