Primers and digital PCR kit for detecting infectious endocarditis pathogens
A kit and reagent technology, applied in the field of detection, can solve the problems of false negatives, inability to take into account internal control channels, and failure to achieve absolute quantification, etc., to achieve good specificity
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Embodiment 1
[0086] Embodiment 1: the detection of staphylococcus aureus, coagulase-negative staphylococcus, enterococcus, streptococcus
[0087] 1. Sample
[0088] (1) Nucleic acid of simulated samples: the gene sequences of various pathogens were synthesized by Tiangen Biological Company. After dissolving the DNA powder, follow the detection panel, mix and dilute 100 times, and freeze 500 times for later use;
[0089] (2) Internal control gene: use the blood / cell / tissue genomic DNA extraction kit to extract the genomic DNA of oral exfoliated cells, dilute to 5ng / μL and freeze for later use;
[0090] (3) Pure cultures of pathogens: pure cultures isolated and cultured from samples of infective endocarditis vegetations from the Microbiology Department of the Laboratory Department of Guangdong Provincial People's Hospital from 2015 to 2021, covering the exception of Rickettsia and Bartonella in this patent (Strictly intracellular parasitism, special culture is required); culture method: a) ...
Embodiment 2
[0121] Example 2: Specificity Verification
[0122] Blast results show that the results described in Description and Scientific Name are all corresponding pathogens, and the comprehensive score (Max Score, Total Score) has the highest score in the retrieved database, and the sequence coverage (Query Cover) reaches 100%. indicating high primer specificity. Figure 6-17 Compare the results for Blast. In addition to blast results, the present invention employs cultures of 12 other common human pathogens (including Salmonella enterica, Enterobacter cloacae, Serratia marcescens, Haemophilus influenzae, Proteus mirabilis, Neisseria meningitidis, Melioides Burkholderia, Mycobacterium tuberculosis, Mycobacterium avium, Nocardiae, Cryptococcus neoformans, Aspergillus fumigatus), the digital PCR system was tested for specificity, and the results were all negative.
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