RAA-Cas12a-Tg: A Nucleic Acid Detection System for Toxoplasma gondii Based on CRISPR-Cas12a Combined with Recombinase-Aided Amplification (RAA).
RAA-Cas12a-Tg: A Nucleic Acid Detection System for Toxoplasma gondii Based on CRISPR-Cas12a Combined with Recombinase-Aided Amplification (RAA).
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DOI：
10.3390/microorganisms9081644
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发表时间：
2021-07-31
期刊：
影响因子：
4.5
通讯作者：
Zhu XQ
中科院分区：
文献类型：
--
作者：
Ma QN;Wang M;Zheng LB;Lin ZQ;Ehsan M;Xiao XX;Zhu XQ
弓形虫病是由细胞内原虫弓形虫引起的一种重要的人畜共患寄生虫病，在世界范围内分布广泛。作为主要传播途径，人类感染可通过从环境(如土壤、水、水果和蔬菜)中摄取弓形虫卵囊而获得。关于环境样本中弓形虫卵囊的检测，开发一种省时、经济、高灵敏度的技术对于监测、预防和控制弓形虫病至关重要。在这项研究中，我们将重组酶辅助扩增(RAA)与CRISPR-Cas12a相结合，开发了一种新的方法，命名为RAA-Cas12a-TG系统。在这里，我们比较了这个针对529个碱基重复元件(529个碱基-RE)的系统与分别针对529个碱基-RE和ITS-1基因的常规聚合酶链式反应，以评估它检测土壤样本中弓形虫卵囊的能力。结果表明，基于Re的529个碱基的RAA-Cas12a-TG体系在体温下能在近1小时内成功检测弓形虫，且比常规的PCR方法更灵敏。该系统灵敏度低至1fM，特异度高。因此，RAA-Cas12a-TG系统提供了一种快速、灵敏、易操作的检测土壤中弓形虫卵囊的方法，这将有助于控制弓形虫在人和动物中的感染。
Toxoplasmosis, caused by the intracellular protozoon Toxoplasma gondii, is a significant parasitic zoonosis with a world-wide distribution. As a main transmission route, human infection can be acquired by the ingestion of T. gondii oocysts from the environment (e.g., soil, water, fruits and vegetables). Regarding the detection of T. gondii oocysts in environmental samples, the development of a time-saving, cost-effective and highly sensitive technique is crucial for the surveillance, prevention and control of toxoplasmosis. In this study, we developed a new method by combining recombinase-aided amplification (RAA) with CRISPR-Cas12a, designated as the RAA-Cas12a-Tg system. Here, we compared this system targeting the 529 bp repeat element (529 bp-RE) with the routine PCR targeting both 529 bp-RE and ITS-1 gene, respectively, to assess its ability to detect T. gondii oocysts in soil samples. Our results indicated that the 529 bp RE-based RAA-Cas12a-Tg system was able to detect T. gondii successfully in nearly an hour at body temperature and was more sensitive than the routine PCR assay. The sensitivity of this system reached as low as 1 fM with high specificity. Thus, RAA-Cas12a-Tg system provided a rapid, sensitive and easily operable method for point-of-care detection of T. gondii oocysts in soil, which will facilitate the control of T. gondii infection in humans and animals.
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DOI：
10.1126/science.aav4294
发表时间：
2018-11-16
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Harrington LB;Burstein D;Chen JS;Paez-Espino D;Ma E;Witte IP;Cofsky JC;Kyrpides NC;Banfield JF;Doudna JA
通讯作者：
Doudna JA
影响因子：
3.2
作者：
Liu Q;Wang ZD;Huang SY;Zhu XQ
通讯作者：
Zhu XQ
DOI：
10.1073/pnas.2010196117
发表时间：
2020-10-13
影响因子：
11.1
作者：
Lee RA;Puig H;Nguyen PQ;Angenent-Mari NM;Donghia NM;McGee JP;Dvorin JD;Klapperich CM;Pollock NR;Collins JJ
通讯作者：
Collins JJ
影响因子：
16.6
作者：
Ding X;Yin K;Li Z;Lalla RV;Ballesteros E;Sfeir MM;Liu C
通讯作者：
Liu C
影响因子：
6.8
作者：
Cong, Wei;Li, Man-Yao;Elsheikha, Hany M.
通讯作者：
Elsheikha, Hany M.