| New Coronavirus pneumonia caused by SARS-Co V-2 has become a major public health event worldwide.Rapid detection of SARS-Co V-2 and early diagnosis of the virus are effective means to control the epidemic.It is found that SARS-Co V-2 can survive for several hours on a smooth surface.Under the special conditions of moderate temperature and humidity in the food cold chain,SARS-Co V-2 can survive for several days,which may lead to the risk of infection.At present,the COVID-19 in China has been effectively controlled.However,due to the continuous deterioration of the epidemic abroad,a large number of imported products(especially food cold chain products)have detected SARS-Co V-2 for many times.Therefore,the rapid and efficient detection of SARS-Co V-2in the outer packaging of food and other items requiring cold chain transportation is an important means to prevent and control the epidemic.At present,nucleic acid detection is still the gold standard for detecting SARS-Co V-2.However,due to the lack of local,timely and convenient detection,the immunological detection method based on antigen can play a great auxiliary role.In this experiment,the N protein with the most conservative gene sequence in SARS-Co V-2 was selected as the immunogen,and the monoclonal antibody against the N protein of SARS-Co V-2 was prepared.Two immunological detection methods,enzyme-linked immunosorbent assay(ELISA)and colloidal gold immunochromatographic strip,were established.The detection conditions of the two methods were optimized and the performance of the experimental methods was evaluated.The two methods can be used to detect SARS-Co V-2 on the surface of food.In this paper,E.coli BL21(DE3)strain containing recombinant N protein gene sequence of SARS-Co V-2 was cultured and induced to express the recombinant N protein.Female balb/c mice were immunized with N protein as immunogen,and the serum titer reached 5.12×10~5times.Two monoclonal antibodies(1F57A6G and 2B39F2B)were screened and cloned.2B39F2B monoclonal antibody with higher titer was selected to establish the follow-up experimental method.N protein indirect competitive ELISA,the optimal condition of this method is:N protein coating amount 0.1μg/well,the dilution ratio of monoclonal antibody is 2×10~6times,0.5%skimmed milk powder was selected as the blocking solution,and the enzyme labeled secondary antibody was diluted by 2×10~5times.The sensitivity and detection limit of the method were 22.16±1.77μg/L and 0.31±0.75μg/L,respectively.The N protein antigen with the concentration of 1.5μg/L was selected as the standard,and the N protein with the final concentration of 5,50,250 and 500μg/L was added.The recovery rates were 95.96%-104.46%,98.62%-106.48%,97.84%-101.45%and99.40%-101.27%respectively,and the standard deviation(SD value)was less than 5%.The optimal conditions of colloidal gold immunochromatographic test strip method were as follows.Sheep anti-mouse secondary antibody(10 mg/m L)was diluted 20 times.N protein coating original(0.5 mg/m L)was diluted 10 times.Nitrocellulose(CN)membrane was selected as NC95 model of Sartorious brand,and the addition amount of N protein monoclonal antibody(1 mg/m L)was 3μL.The addition amount of potassium carbonate(0.2 mol/L)was 5μL.The loading buffer was PBS of p H 7.4.The visual detection limit(v LOD)and critical value were 62μg/L and 500μg/L with good specificity,stability and repeatability.The results of the recovery test of the negative samples showed that the results were consistent with those of the commercial test strips,so it had good accuracy.The test strip method can get the results in about 10 minutes,and can be used as an important means of early screening.The two rapid detection methods of novel coronavirus antigen established in this study have a good application prospect for the early detection,prevention and control of SARS-COV-2. |
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