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METHOD FOR DETERMINING FALSE NEGATIVE AND FALSE POSITIVE REACTIONS FOR TARGET PATHOGEN GENE DETECTION BASED ON INTERNATIONAL STANDARD REAL-TIME RT-PCR OF VIRAL HAEMORRHAGIC SEPTICAEMIA
METHOD FOR DETERMINING FALSE NEGATIVE AND FALSE POSITIVE REACTIONS FOR TARGET PATHOGEN GENE DETECTION BASED ON INTERNATIONAL STANDARD REAL-TIME RT-PCR OF VIRAL HAEMORRHAGIC SEPTICAEMIA
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机译：基于国际标准实时RT-PCR的毒性出血性遗产症的靶病原体基因检测确定假阴性和假阳性反应的方法
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摘要
The present invention relates to a method for discriminating false negatives and false positives that may appear in the detection of a pathogen gene of a target disease based on a real-time RT-PCR method for viral hemorrhagic sepsis, and more specifically, (1) Jonstrup VHS rRT-PCR Synthesizing by linking and synthesizing a nucleotide sequence according to the law and a gene for a nucleotide sequence for detecting a pathogen gene of a target disease and determining false positives; (2) Dilute the synthesized gene stepwise and use each dilution as a template, but perform the Jonstrup VHS rRT-PCR method and the rRT-PCR reaction for detecting the pathogen gene of the target disease, respectively, and compare the detection sensitivity for the two reactions. Selecting a synthetic nucleotide sequence for detection of a disease pathogen gene; (3) extracting synthetic recombinant plasmid DNA by cloning the gene synthesized in step (1) into a plasmid DNA vector; (4) Using the extracted synthetic recombinant plasmid DNA as a template, primers and probes according to the synthetic nucleotide sequence for detection of the pathogen gene of the target disease selected in step (2), for VHSV detection according to the Jonstrup VHS rRT-PCR method Incorporating primers and probes, primers and probes for detection of plasmid vector DNA into a real-time PCR cocktail preparation, and performing multiple real-time RT-PCR; (5) From the results of the multiple real-time RT-PCR, by comparing the detection sensitivity of VHSV and the detection sensitivity of the pathogen gene of the target disease, verifying the sensitivity of the pathogen gene detection method of the target disease, false by using the low sensitivity detection method. Preventing negative reactions; (6) using the synthetic recombinant plasmid DNA as a positive control, when diagnosing the target disease, confirming the success of multiple real-time PCR experiments for detection of the target disease pathogen gene without direct use of the target disease pathogen; (7) When diagnosing the target disease, if the pathogen of the target disease is an RNA virus, the real-time to determine the success of RNA extraction and cDNA synthesis by setting the eukaryotic common gene detection as an internal control. Incorporating a eukaryotic common gene detection primer and a probe into the PCR cocktail preparation, verifying the RNA extraction and cDNA synthesis experimental process through confirmation of the internal control fluorescence reaction; And (8) at the time of diagnosis of the target disease, determining false negatives and false positives by confirming multiple fluorescence reaction results. According to the method for determining false negatives and false positives that may appear in the detection of the pathogen gene of the target disease based on the real-time RT-PCR method for viral hemorrhagic sepsis proposed in the present invention, Jonstrup standardized on a structurally stable plasmid DNA vector. Synthetic recombinant plasmid DNA is extracted by cloning a synthetic gene containing the base sequence according to the VHS rRT-PCR method and the base sequence for detecting the target disease at the same time, and comparing the detection sensitivity of real-time RT-PCR performed using this, the target disease By verifying the effectiveness of the detection sensitivity of the pathogen gene and adjusting it, it is possible to establish a method for detecting a high-sensitivity gene for the target disease within a short period of time. In addition, according to the method for determining false negatives and false positives that may appear in the detection of the pathogen gene of the target disease based on the real-time RT-PCR method for viral hemorrhagic sepsis proposed in the present invention, whether the plasmid vector gene is detected and amplified By comparing the levels, it is possible to determine whether or not the synthetic recombinant plasmid DNA used as a positive control is contaminated. In addition, according to the method for determining false negatives and false positives that may appear in the detection of the pathogen gene of the target disease based on the real-time RT-PCR method for viral hemorrhagic sepsis proposed in the present invention, the detection of the 18s rRNA gene is determined. In comparison, it is possible to discriminate against contamination that may occur due to mold in the laboratory or carelessness of the experimenter, and comprehensively control false positive and false negative responses to the diagnostic sample of the target disease.
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