A sample nucleic acid detection kit, a sample nucleic acid detection reagent and an application for a sample nucleic acid detection reagent

Abstract

The invention provides a detection sample nucleic acid reagent, method and kit. Nucleic acid in a sample is extracted by adopting highly basic nucleic acid extract (pH value is 10.5-12.5), then extracted nucleic acid is directly added into a PCR reaction solution (pH is 8.0-9.0) to perform neutralization, and nucleic acid amplification and detection are performed simultaneously. The nucleic acid in different samples such as blood, blood plasma, whole blood, genital meatus discharge, sputum or urine can be detected. Through performing the neutralization on the extracted sample nucleic acid in the PCR reaction solution, rather than performing the neutralization during extracting the nucleic acid, the nucleic acid extraction efficiency and the PCR amplification efficiency can be effectively improved, and accuracy and sensitivity of the detection for the sample nucleic acid are substantially improved.

Description

technical field [0001] The invention belongs to the field of biological science and biotechnology, in particular to a reagent, method and kit for detecting sample nucleic acid. Background technique [0002] With the rapid development of molecular biology technology, it has penetrated into various aspects of biology, medicine, botany, genetics and zoology. As a major breakthrough in molecular biology technology, polymerase chain reaction (PCR) technology has the characteristics of high sensitivity, high specificity, time saving and rapidity, and has been widely used in human and animal disease diagnosis (such as prenatal diagnosis). , newborn screening and genetic metabolic disease detection), forensic detection, kinship analysis, seed purity identification, molecular marker-assisted breeding, gene mapping and genetically modified organism detection, etc. [0003] The use of PCR technology for detection mainly involves three aspects: nucleic acid extraction, nucleic acid amp...

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Problems solved by technology

Similarly, when using this method to extract nucleic acids in biological samples, it is necessary to manually add neutralizing solution for neutralization, and the operation steps are somewhat cumbersome. If the operator forgets to add it, the PCR amplification efficiency will be reduced and the detection results will be affected.

[0008] Regardless of whether the Chelex-100 boiling method or the alkaline lysis method is used, when using existing technologies to extract and detect nucleic acids in samples, there will always be a problem: taking the detection of HBV DNA that may exist in clinical blood or plasma samples as an example, using strong The alkaline nucleic acid extraction solution extracts the nucleic acid in clinical blood or plasma samples to obtain the extracted nucleic acid solution, and then adds 2 to 5 μl of the extracted nucleic acid solution to about 35 μl of the PCR reaction solution for mixing. This is due to the addition of The volume of the extracted nucleic acid solution is small, and has little effect on the mixed PCR reaction solution, but when the volume of the extracted nucleic acid solution added is large, such as adding 20 μl of the extracted nucleic acid solution to 20 μl PCR When mixing in the reaction solution, although the total volume is still 40 μl, the volume ratio of the extracted nucleic acid solution increases significantly, which will affect the mixed PCR reaction solution, resulting in a high pH value, which will affect the subsequent PCR amplification. Increase reaction efficiency, reduce detection sensitivity and accuracy of sample nucleic acid

[0009] In addition, after using conventional weak alkaline nucleic acid lysate to extract nucleic acid in the sample, when the volume of the extracted nucleic acid solution added to the PCR reaction solution is small (usually 2-5 μl), although the nucleic acid in the sample can be detected Nucleic acid, but the detection sensitivity is low; however, when the volume of the extracted nucleic acid solution added is large, such as adding 20 μl of the extracted nucleic acid solution to the 20 μl PCR reaction solution for mixing, at this time due to weak alkaline nucleic acid lysate extraction The efficiency of the sample nucleic acid is poor, and the large volume of nucleic acid solution added is likely to contain more impurities and PCR amplification reaction inhibitors, which will often cause the PCR amplification reaction to fail to proceed normally, or even if the PCR amplification can be performed Reaction, but will reduce the detection sensitivity and accuracy of sample nucleic acid

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