| Background and objective: Toxoplasma gondii infection causes toxoplasmosis,which affects any organ in the body.Toxoplasma gondii infection is usually asymptomatic in people with normal immune function,and in rare cases,flu-like illnesses may occur;however,in people with weakened immune function,such as AIDS patients,it can cause toxoplasma eye disease,meningoencephalitis,liver necrosis,and other illnesses,and can be fatal in severe cases.For women during pregnancy,miscarriage,premature birth,and abnormal fetus may occur.According to epidemiological data,porcine toxoplasmosis is widespread in China,and pigs infected with Toxoplasma gondii will show symptoms such as retention fever,diarrhea,and severe loss of appetite,and porcine Toxoplasma gondii infection is explosive in farms,which can bring unacceptable economic losses if not detected and intervened in time.Therefore,the establishment of a rapid and accurate diagnosis method for Toxoplasma gondii is essential for the prevention and control of Toxoplasma gondii and eugenics.There are numerous testing methods for Toxoplasma gondii infection;the more widely used in hospitals is antibody testing,but because antibody testing has a window period and cannot be avoided.several other tests that can improve the accuracy of the results,such as PCR and QPCR,require expensive and precise instruments and cannot be popularized in some remote areas with limited medical conditions.In recent years,RAA amplification technology and CRISPR/Cas12 a highly specific targeted detection technology have emerged,which can be performed at 37 ℃,and the combination of the two can circumvent the disadvantages of the above assays.This method is sensitive,specific,simple and easy to operate,and can be used for POCT.It has been applied to the detection of parasites and has a broad prospect in the field of molecular detection.The aim of this paper is to establish an efficient method for the detection of Toxoplasma gondii infection in combination with RAA and CRISPR/Cas12 a,and to test the feasibility of this method by standards and modeling samples.Methods: A method for RAA combined with CRISPR/Cas12 a was established to detect Toxoplasma gondii DNA,and the ratio of protein to cr RNA was optimized to minimize the cost.The optimized system was used to test the sensitivity of plasmid standards and Toxoplasma gondii DNA.four T.gondii strains,RH,Pru,wh3,and wh6,were selected for universal detection,and the specificity of protozoa with similar T.gondii habits was assessed.In order to verify the application value of this method,we performed blood DNA detection in the established mice model of T.gondii infection.Considering POCT,we apply the strip to the result reading section,load the detection system onto the strip,and observe the results directly.Results: The sensitivity of the standard was evaluated by the established RAA combined with CRISPR/Cas12 a method for the detection of T.gondii DNA,and the detection limit of both gene loci was 1.5 cp/μl,which was consistent with the highly sensitive nested PCR results.For the detection of Toxoplasma gondii DNA,the B1 gene detected 1tachyzoite/response and the 529 RE detected 0.1 tachyzoite/response.This method was suitable for other strains except RH,and there was no cross-reaction with other protozoal DNAs with similar habits.For the Toxoplasma gondii-infected mice model,all blood samples from 1000 RH tachyzoites infected mice were detected as positive for Toxoplasma gondii on day 1,day 3,and day 5.The test results can be observed not only by fluorescence reading instrument,but also directly under UV light and by test strip reading.Conclusion:In this study,we established a time-saving,sensitive,single-copy detection method for Toxoplasma gondii DNA,which can target all types of toxoplasmosis caused by Toxoplasma infection and has the potential to be an alternative tool for on-site detection of Toxoplasma gondii. |
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