Dao Thi Huyen, Julien Reboud, Dao Thanh Quyen, Jonathan M Cooper, Thirumalaisamy P Velavan, Ngo Tat Trung, Le Huu Song
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LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. The limit of detection (LOD) was established with serial dilutions of the target N. meningitidis DNA and calculated by Probit regression analysis.</p><p><strong>Results: </strong>Six LAMP assay-specific primers were developed targeting the ctrA gene of N. meningitidis, which is conserved in all meningococcal serogroups. The LAMP primers did not amplify DNA from other bacterial DNA tested, showing 100% specificity. The use of 0.4 M betaine increased the sensitivity and stability of the reaction. LAMP-CRISPR/Cas detected meningococcal serogroups (B, C, W). The assay showed no cross-reactivity and was specific for N. meningitidis. The LOD was 74 (95% CI: 47-311) N. meningitidis copies. The LAMP-CRISPR/Cas performed well compared to the gold standard. In the 139 samples from suspected patients, the sensitivity and specificity of the test were 91% and 99% respectively.</p><p><strong>Conclusion: </strong>This developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.</p>","PeriodicalId":8052,"journal":{"name":"Annals of Clinical Microbiology and Antimicrobials","volume":"23 1","pages":"28"},"PeriodicalIF":4.6000,"publicationDate":"2024-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10981803/pdf/","citationCount":"0","resultStr":"{\"title\":\"An isothermal CRISPR- based lateral flow assay for detection of Neisseria meningitidis.\",\"authors\":\"Dao Thi Huyen, Julien Reboud, Dao Thanh Quyen, Jonathan M Cooper, Thirumalaisamy P Velavan, Ngo Tat Trung, Le Huu Song\",\"doi\":\"10.1186/s12941-024-00688-1\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Neisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time consuming. This study aimed to combine the sensitivity of loop-mediated isothermal nucleic acid amplification (LAMP) with the specificity of CRISPR/Cas12a cleavage to demonstrate a reliable diagnostic assay for rapid detection of N. meningitidis.</p><p><strong>Methods: </strong>A total of n = 139 samples were collected from patients with suspected meningococcal disease and were used for evaluation. The extracted DNA was subjected to qualitative real-time PCR, targeting capsular transporter gene (ctrA) of N. meningitidis. LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. 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引用次数: 0
摘要
背景：脑膜炎奈瑟菌可导致危及生命的脑膜炎球菌性脑膜炎和脑膜炎球菌血症。从脑脊液/血液培养中获得旧标准微生物学结果非常耗时。本研究旨在将环介导等温核酸扩增（LAMP）的灵敏性与CRISPR/Cas12a裂解的特异性相结合，展示一种可靠的快速检测脑膜炎球菌的诊断方法：方法：从疑似脑膜炎球菌疾病患者身上共采集了 n = 139 份样本用于评估。对提取的 DNA 进行定性实时 PCR 检测，目标是脑膜炎球菌的荚膜转运体基因（ctrA）。设计了同样针对ctrA的LAMP特异引物对，并对LAMP产物进行CRISPR/Cas12裂解反应。LAMP-CRISPR/Cas 的灵敏度、特异性、阳性预测值 (PPV) 和阴性预测值 (NPV) 与实时 PCR 检测进行了比较。利用目标脑膜炎球菌 DNA 的系列稀释液确定了检测限（LOD），并通过 Probit 回归分析进行了计算：结果：针对脑膜炎球菌的ctrA基因开发了六种LAMP检测特异引物，该基因在所有脑膜炎球菌血清群中都是保守的。LAMP 引物不会扩增其他细菌 DNA，特异性达到 100%。使用 0.4 M 甜菜碱提高了反应的灵敏度和稳定性。LAMP-CRISPR/Cas 检测脑膜炎球菌血清群（B、C、W）。该检测方法没有交叉反应，对脑膜炎球菌具有特异性。LOD 为 74（95% CI：47-311）个脑膜炎球菌拷贝。与金标准相比，LAMP-CRISPR/Cas 的性能良好。在 139 份疑似患者样本中，检测的灵敏度和特异性分别为 91% 和 99%：结论：这一经过开发和优化的方法可作为现有金标准的补充，用于及时诊断脑膜炎球菌性脑膜炎和脑膜炎球菌血症。
An isothermal CRISPR- based lateral flow assay for detection of Neisseria meningitidis.
Background: Neisseria meningitidis can cause life-threatening meningococcal meningitis and meningococcemia. Old standard microbiological results from CSF/blood cultures are time consuming. This study aimed to combine the sensitivity of loop-mediated isothermal nucleic acid amplification (LAMP) with the specificity of CRISPR/Cas12a cleavage to demonstrate a reliable diagnostic assay for rapid detection of N. meningitidis.
Methods: A total of n = 139 samples were collected from patients with suspected meningococcal disease and were used for evaluation. The extracted DNA was subjected to qualitative real-time PCR, targeting capsular transporter gene (ctrA) of N. meningitidis. LAMP-specific primer pairs, also targeting the ctrA, were designed and the LAMP products were subjected to CRISPR/Cas12 cleavage reaction. the readout was on a lateral flow strip. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of LAMP-CRISPR/Cas was compared with real-time PCR assays. The limit of detection (LOD) was established with serial dilutions of the target N. meningitidis DNA and calculated by Probit regression analysis.
Results: Six LAMP assay-specific primers were developed targeting the ctrA gene of N. meningitidis, which is conserved in all meningococcal serogroups. The LAMP primers did not amplify DNA from other bacterial DNA tested, showing 100% specificity. The use of 0.4 M betaine increased the sensitivity and stability of the reaction. LAMP-CRISPR/Cas detected meningococcal serogroups (B, C, W). The assay showed no cross-reactivity and was specific for N. meningitidis. The LOD was 74 (95% CI: 47-311) N. meningitidis copies. The LAMP-CRISPR/Cas performed well compared to the gold standard. In the 139 samples from suspected patients, the sensitivity and specificity of the test were 91% and 99% respectively.
Conclusion: This developed and optimized method can complement for the available gold standard for the timely diagnosis of meningococcal meningitis and meningococcemia.
期刊介绍：
Annals of Clinical Microbiology and Antimicrobials considers good quality, novel and international research of more than regional relevance. Research must include epidemiological and/or clinical information about isolates, and the journal covers the clinical microbiology of bacteria, viruses and fungi, as well as antimicrobial treatment of infectious diseases.
Annals of Clinical Microbiology and Antimicrobials is an open access, peer-reviewed journal focusing on information concerning clinical microbiology, infectious diseases and antimicrobials. The management of infectious disease is dependent on correct diagnosis and appropriate antimicrobial treatment, and with this in mind, the journal aims to improve the communication between laboratory and clinical science in the field of clinical microbiology and antimicrobial treatment. Furthermore, the journal has no restrictions on space or access; this ensures that the journal can reach the widest possible audience.
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