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

Inactive Publication Date: 2012-04-11
JIANGSU UNIV
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Problems solved by technology

The current routine detection method for Streptococcus pneumoniae, Haemophilus influenzae type b, and Mycobacterium tuberculosis complex is based on the culture method, which is time-consuming and has low sensitivity
There is currently no multiplex PCR report that can simultaneously detect Streptococcus pneumoniae, Haemophilus influenzae type b and Mycobacterium tuberculosis complex

Method used

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  • Multiple landing PCR(Polymerase Chain Reaction) detection kit and detection method for pathogenic bacteria of lower respiratory tract
  • Multiple landing PCR(Polymerase Chain Reaction) detection kit and detection method for pathogenic bacteria of lower respiratory tract

Examples

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Embodiment 1

[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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Abstract

The invention designs specific primers for streptococcus pneumoniae, haemophilus influenzae type b and mycobacterium tuberculosis complex, and establishes a multiple landing PCR(Polymerase Chain Reaction) detection kit and a detection method for simultaneously detecting the bacteria, adopting agarose gel electrophoresis for detecting PCR products. The kit comprises a 10*PCR buffer solution, MgCl2, dNTP, TaqDNA polymerase, BSA(Bovine Serum Albumin), positive control DNAs of streptococcus pneumoniae, haemophilus influenzae type b and mycobacterium tuberculosis complex, a streptococcus pneumoniae primer, a haemophilus influenzae type b primer and a mycobacterium tuberculosis complex primer. The kit can simultaneously quickly detect streptococcus pneumoniae, haemophilus influenzae type b and mycobacterium tuberculosis complex in a lower respiratory tract sample. The multiple landing PCR detection kit and detection method established by the invention are quick, simple, specific and sensitive, and can be used for rapid diagnosis and epidemiological investigation of infection of streptococcus pneumoniae, haemophilus influenzae type b and mycobacterium tuberculosis complex.

Description

technical field [0001] The present invention relates to the technical field of molecular diagnosis of lower respiratory tract infectious diseases, in particular to multiple landing PCR detection method in lower respiratory tract specimens of Streptococcus pneumoniae ( Streptococcus pneumoniae ), Haemophilus influenzae type b ( Haemophilus influenzae type b ) and Mycobacterium tuberculosis complex ( Mycobacterium tuberculosis complex ) detection application. Background technique [0002] Streptococcus pneumoniae and Haemophilus influenzae type b are the main pathogens causing community-acquired pneumonia. Streptococcus pneumoniae and Haemophilus influenzae type b vaccines have not yet been included in my country's first-class immunization program. The Mycobacterium tuberculosis complex is a group of mycobacteria that cause tuberculosis, including Mycobacterium tuberculosis ( Mycobacterium tuberculosis ), Mycobacterium bovis ( Mycobacterium bovis ), BCG ( Mycobacte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/14C12Q1/04C12R1/46C12R1/32C12R1/21
Inventor 邵世和罗欲承徐会娟杜蓬段秀杰侯艳娇潘红
Owner JIANGSU UNIV
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