Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic Gram-negative pathogen that may cause infections to immunocompromised patients. However, sensitive and reliable analysis of P. aeruginosa remains a huge challenge. In this method, target recognition assists the formation of a self-primer and initiates single-stranded chain production. The produced single-stranded DNA chain is identified by CRISPR-Cas12a, and consequently, the trans-cleavage activity of the Cas12a enzyme is activated to parallelly digest Ag⁺ aptamer sequences that are chelated with silver ions (Ag⁺). The released Ag⁺ reacted with 3,3′,5,5′-tetramethylbenzidine (TMB) for coloring. Compared with the traditional color developing strategies, which mainly rely on the DNA hybridization, the color developing strategy in this approach exhibits a higher efficiency due to the robust trans-cleavage activity of the Cas12a enzyme. Consequently, the method shows a low limit of detection of a wide detection of 5 orders of magnitudes and a low limit of detection of 21 cfu/mL, holding a promising prospect in early diagnosis of infections. Herein, we develop a sensitive and reliable method for direct and colorimetric detection of P. aeruginosa by integrating self-primer-assisted chain production and CRISPR-Cas12a-based color reaction and believe that the established approach will facilitate the development of bacteria-analyzing sensors.

中文翻译：

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通过自引物辅助扩链和基于 CRISPR-Cas12a 的显色反应对铜绿假单胞菌进行灵敏、直接的分析

铜绿假单胞菌（P. aeruginosa）是一种常见的机会性革兰氏阴性病原体，可能导致免疫功能低下患者感染。然而，对铜绿假单胞菌的灵敏且可靠的分析仍然是一个巨大的挑战。在该方法中，目标识别有助于自引物的形成并启动单链生产。产生的单链DNA链被CRISPR-Cas12a识别，从而激活Cas12a酶的反式切割活性以平行消化与银离子（Ag ⁺）螯合的Ag ^{+适体序列。}释放出的Ag ⁺与3,3',5,5'-四甲基联苯胺(TMB)反应显色。与主要依靠DNA杂交的传统显色策略相比，由于Cas12a酶强大的反式切割活性，该方法的显色策略表现出更高的效率。因此，该方法显示出5个数量级的宽检测低限和21 cfu/mL的检测低限，在感染的早期诊断中具有广阔的前景。在此，我们通过整合自引物辅助链生产和基于 CRISPR-Cas12a 的显色反应，开发了一种灵敏可靠的方法，用于直接比色检测铜绿假单胞菌，并相信所建立的方法将促进细菌分析传感器的开发。