Multiplex PCR and Nanopore Sequencing of Genes Associated with Antimicrobial Resistance in Neisseria gonorrhoeae Directly from Clinical Samples
Multiplex PCR and Nanopore Sequencing of Genes Associated with Antimicrobial Resistance in Neisseria gonorrhoeae Directly from Clinical Samples
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DOI：
10.1093/clinchem/hvaa306
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发表时间：
2021-04-01
影响因子：
9.3
通讯作者：
Peng, Junping
中科院分区：
文献类型：
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作者：
Zhang, Chi;Xiu, Leshan;Peng, Junping
背景：淋病奈瑟菌的抗微生物药物耐药性（AMR）已在世界范围内蔓延。需要快速全面的方法来准确描述淋病奈瑟菌AMR谱。建立了一种基于多重扩增子测序的方法，可同时对淋病奈瑟菌临床样本中与AMR相关的13个基因进行测序。方法：以9株淋病奈瑟菌为研究对象，建立方法并进行验证。将11份尿道拭子及其相应的培养分离物配对，以确定方法的准确性。制备不同稀释度的模拟样品，以确定该方法的灵敏度。五种非淋球菌奈瑟菌菌株和24N。采用淋病阴性临床样本评价交叉反应性。最后，将该方法应用于64个临床样本，以评估其性能。结果：以Sanger测序为参考方法，扩增子测序恢复的序列碱基精度达99.5%以上，AMR位点鉴定正确。检出限(LOD) < 31拷贝/反应。未观察到明显的交叉反应。此外，从64份临床样本中成功回收了靶基因，其中包括9份尿液，表明该方法可用于不同类型的样本。对于临床样品，可在7小时40分钟至10小时40分钟的时间范围内获得结果，而对于分离株，周转时间约短2小时。结论：该方法具有灵敏度和准确性高的优点，可作为一种通用、便捷的无培养诊断方法。
BACKGROUND: Antimicrobial resistance (AMR) of Neisseria gonorrhoeae has spread worldwide. Rapid and comprehensive methods are needed to describe N. gonorrhoeae AMR profiles accurately. A method based on multiplex amplicon sequencing was developed to simultaneously sequence 13 genes related to AMR in N. gonorrhoeae directly from clinical samples.METHODS: Nine N. gonorrhoeae strains were used for the establishment and validation of the method. Eleven urethral swabs and their corresponding cultured isolates were matched as pairs to determine the accuracy of the method. Mock samples with different dilutions were prepared to determine the sensitivity of the method. Five nongonococcal Neisseria strains and 24N. gonorrhoeae negative clinical samples were used to evaluate the cross-reactivity. Finally, the method was applied to 64 clinical samples to assess its performance.RESULTS: Using Sanger sequencing as a reference method, sequences recovered from amplicon sequencing had a base accuracy of over 99.5% and the AMR sites were correctly identified. The limit of detection (LOD) was lower than 31 copies/reaction. No significant cross-reactivity was observed. Furthermore, target genes were successfully recovered from 64 clinical samples including 9 urines, demonstrating this method could be used in different types of samples. For clinical samples, the results can be obtained within a time frame of 7 h 40 min to 10 h 40 min, while for isolates, the turnaround time was approximately 2 h shorter.CONCLUSIONS: This method can serve as a versatile and convenient culture-free diagnostic method with the advantages of high sensitivity and accuracy.