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Detection kit capable of directly amplifying virus sample containing SARS CoV-2 and detection method thereof
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A detection kit and direct technology, applied in the field of biomedicine, can solve the problems of inability to directly amplify detection, invasive collection, low detection sensitivity, etc., and achieve the effect of shortening the sample processing process, reducing the time used, and simple operation
Pending Publication Date: 2021-07-23
SUZHOU SYM BIO LIFESCI CO LTD
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[0005] At present, the kits that use the new coronavirusRNA as the detection object need to use a specific kit to extract the RNAnucleic acid from the viral RNA template, which cannot be directly amplified for detection; although the kits that use the new coronavirusantigenantibody as the detection object can Directly use blood for detection, but the detection sensitivity is much lower than that of kits that use RNA as the detection object, and it is collected with trauma
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Embodiment 1
[0063] Take 10 cases of nasopharyngeal swabs from patients who are negative for the new coronavirus, put them into a sample tube containing 400 μL of sample treatment solution, shake vigorously for 30 seconds, discard the swabs, shake vigorously again for 30 seconds, and take 5 μL for detection by the above-mentioned direct amplification method. The test results were all negative.
[0064] Among them, the PCR instrument used is ABI 7500, and the internal standard amplification curve is as follows: figure 1 As shown, neither N nor ORF1ab was detected in the sample to be tested.
Embodiment 2
[0066] Take 10 throat swabs from patients who are negative for the novel coronavirus, put them into a sample tube containing 400 μL of sample treatment solution, shake vigorously for 30 seconds, discard the swabs, shake vigorously again for 30 seconds, and take 5 μL for detection by the above-mentioned direct amplification method. The results were all negative.
[0067] Among them, the PCR instrument used is ABI 7500, and the internal standard amplification curve is as follows: figure 2 As shown, N and ORF1ab genes were not detected in the samples to be tested.
Embodiment 3
[0069] The sample treatment solution of negative nasopharyngeal swabs was mixed and mixed with the new coronavirus inactivated virus culture (NR-52287) for treatment, respectively prepared into different concentrations, 3 times the concentration and doubling dilution, used for preliminary exploration of direct amplification of samples The detection limit of the incremental method, and finally determine the minimum detection limit through 20 repeated tests, the positive rate of 20 repeated tests must be ≥ 95%, and the confirmed detection limit, LoD, is defined as 1.1E+04cp / mL.
[0070] Wherein, the PCR instrument adopted is QuantStudio Dx 96 of Thermo.
[0071] The detection limit data of 3 times concentration doubling dilution is as follows:
[0072] Table 1
[0073]
[0074]
[0075] Among them, when the Ct of any gene of N and ORF1ab is ≤ 42, it is defined as the sample is positive for SARS-CoV-2.
[0076] 20 times of repeated test verification detection limit data a...
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Abstract
The invention relates to a detection kit capable of directly amplifying a virus sample containing SARS CoV-2. The detection kit comprises a sample treatment solution, a hot start anti-inhibition DNApolymerasemixed solution, a reaction reagent A, a reaction reagent B, a reaction reagent C, a negative control, a positive control and an internal standard; the sample treatment solution comprises EDTA (ethylenediamine tetraacetic acid), SDS (sodiumdodecyl sulfate) and a detergent; the reaction reagent A comprises Tris, ammoniumsulfate, magnesiumchloride and dNTPs; the reaction reagent B comprises a primer probe set as shown in SEQ ID NO.1-6; the reaction reagent C comprises BSA (bovine serum albumin), gelatin and Brij; and the hot start anti-inhibition DNApolymerasemixed solution comprises Taq, MMLV, UNG and RNase Inhibitor. The invention also relates to a direct amplification detection method adopting the detection kit. The method is simple to operate and does not need professional and tedious extraction reagents and extraction instruments, the sample can be rapidly lysed and be directly used for detection, the sample treatment flow is effectively shortened, the PCR detection time is reduced, and a nucleic acid detection result is rapidly provided.
Description
technical field [0001] The invention relates to the field of biomedicine, in particular to a detection kit capable of directly amplifying a virus sample containing SARS CoV-2 and a detection method thereof. Background technique [0002] The clinical symptoms of 2019-nCoV-infected patients are mainly fever, fatigue, and dry cough. Upper respiratory symptoms such as nasal congestion and runny nose are rare, and hypoxic and hypoxic states will occur. Some patients developed dyspnea more than a week later, and severe cases rapidly progressed to acute respiratory distress syndrome, septic shock, difficult-to-correct metabolic acidosis and coagulation dysfunction. [0003] There are currently a large number of new coronavirus detection kits on the market, mainly including fluorescence-PCR method, combined probe-anchored polymerization sequencing method, constant temperature amplification chip method, magnetic particle chemiluminescence method, hybrid capture immunofluorescence m...
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