RPB0570

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
Toxoplasma gondii Toxoplasma gondii 5811 Eucoccidiorida Sarcocystidae Toxoplasma Toxoplasma gondii Eukaryota

Primer Description

Primer Name Sequence(5'-3') Length(bp) Primer Final Concentration(μM） GC Content（%） Predicted Melting Temperature(℃） Molecular Weight(g/moles) Positions in GenBank accession number
B1-RPA-F TGAGGTCATATCGTCCCATGAAGTCGACCACC 32 420 nM 53.13 66.09 9769.4 \
B1-RPA-R CAAGAATGTT GCATTCTTCA GCCGTCTTGT GG 32 420 nM 46.88 63.28 9821.42 \
B1-RPA-exoP ATTCAAGGGAAGAGATCCAGCAGATCTCGTTCG(FAM-dT)(THF)(BHQ1-dT)ATTCGAGACAAGAG-C3 spacer 47 120 nM 46.81 68.05 14569.53 \

Gene Description

Target Gene GenBank ID
B1 gene AF179871.1

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
The visual signal readout can be easily realized by a fluorometer or lateral-flow strip. A portable suitcase containing the minimum equipment and lyophilized reagents was adopted for the rapid determination of T. gondii in heavily polluted landfill leachate. This system presents rapidness, robustness and on-site features for the detection of nucleic acids of the parasite, making it a promising tool for field applications in remote areas. RPA\CRISPR\Cas12a NCBI 25 min 37 °C CRISPR\Cas12a 3.3 copies/μL \ \

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2022 RPA\CRISPR\Cas12a-Based On-Site and Rapid Nucleic Acid Detection of Toxoplasma gondii in the Environment Rong Lei,Limei Li,Pinshan Wu,Xinyu Fei,Yuting Zhang,Jingyi Wang,Di Zhang,Qingfang Zhang,Na Yang,Xinyi Wang ACS synthetic biology 35471824 10.1021/acssynbio.1c00620

RPA\CRISPR\Cas12a-Based On-Site and Rapid Nucleic Acid Detection of Toxoplasma gondii in the Environment

Author(s):

Rong Lei,Limei Li,Pinshan Wu,Xinyu Fei,Yuting Zhang,Jingyi Wang,Di Zhang,Qingfang Zhang,Na Yang,Xinyi Wang

Journal:

ACS synthetic biology

Year:

2022

Abstract:

Toxoplasma gondii is an opportunistic pathogen widely distributed within the world, poses a huge threat to human health, and causes significant economic losses to the livestock industry. Herein, we developed a portable one-pot detection of T. gondii by combining recombinase polymerase amplification (RPA) and a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. A glass microfiber filter device used for the first step can efficiently extract T. gondii from low-concentration samples. The lyophilized RPA reagents and Cas12a/crRNA reagents are prestored in one Eppendorf tube, and both reactions can be performed on a low-cost thermal controller (∼37 °C), avoiding the drawbacks of the step-by-step addition of components. The developed RPA/CRISPR/Cas12a system exhibits a high selectivity toward the B1 gene amplicon of T. gondii over other parasites with a limit of detection of 3.3 copies/μL. The visual signal readout can be easily realized by a fluorometer or lateral-flow strip. A portable suitcase containing the minimum equipment and lyophilized reagents was adopted for the rapid determination of T. gondii in heavily polluted landfill leachate. This system presents rapidness, robustness and on-site features for the detection of nucleic acids of the parasite, making it a promising tool for field applications in remote areas.