Multiple landing PCR(Polymerase Chain Reaction) detection kit and detection method for pathogenic bacteria of lower respiratory tract
A technology for pathogenic bacteria and respiratory tract, which is applied to the multiple-drop PCR detection kit and detection field of pathogenic bacteria in the lower respiratory tract, can solve the problems of low sensitivity and time-consuming operation, and achieve the effect of high sensitivity
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[0043] Pretreatment of sputum specimens: the sputum specimens were washed twice with normal saline, added with an equal amount of 1% trypsin (prepared and used on the same day) and digested at 37°C for 90 min.
[0044] Bacterial DNA extraction from sputum specimens: 1.5 ml of digested sputum specimens were used to extract bacterial DNA from sputum specimens with Takara minibest Bacterial Genome Extraction Kit (Takara, Dalian Baosheng). The extracted DNA was directly used for PCR amplification or stored at -20°C for future use.
[0045] 25 μl total PCR amplification system, add 11.55 μl double distilled water, 2.5 μl buffer (10×PCR), 0.25 μl BSA (10 mg / ml), primer (10 μmol / L) Sp_cpsA in 200 μl PCR tube -F, Sp_cpsA-R, Hib_cap-F, Hib_cap-R, TB_IS6110-F, TB_IS6110-R 1 μl each, 2 μl MgCl 2 (25 mM), 0.5 μl dNTP (10 mM), 0.2 μl Taq DNA polymerase (5U / μl), 2 μl extracted sputum sample DNA. Streptococcus pneumoniae, Haemophilus influenzae type b, and Mycobacterium tuberculosis po...
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