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Method for detecting infectious disease pathogens and kit

A technology for infectious diseases and pathogens, applied in the field of molecular biology, can solve the problem of not being able to detect multiple infectious disease pathogens at the same time, and achieve the effects of fast detection, improved specificity, and mild reaction conditions

Active Publication Date: 2014-07-16
HAI KANG LIFE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The purpose of the present invention is to overcome the defect that the prior art cannot simultaneously detect multiple infectious disease pathogens, and provide a method for detecting infectious disease pathogens that may exist in biological samples, including:

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Design and prepare primers and probe sequences.

[0069] According to the NCBI search, the specific gene sequences of Chlamydia pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae and Legionella pneumophila were selected, which are respectively:

[0070] The outer membrane protein A gene sequence of Chlamydia pneumoniae, whose GENEBANK accession number is AY555078, selects the highly conserved part of the sequence as the target nucleic acid sequence, and the target sequence selected in the present invention is the outer membrane protein A gene 660-893, and its nucleotide sequence is as SEQ ID As shown in NO.1;

[0071] Haemophilus influenzae outer membrane protein P6 gene sequence, GENEBANK accession number is M19391, the target nucleic acid sequence selected in the present invention is 173-372 of the outer membrane protein P6 gene, its nucleotide sequence is shown in SEQID NO.2;

[0072] Mycoplasma pneumoniae adhesion protein P1 gene se...

Embodiment 2

[0084] The kits described in the present invention are prepared. Composed of:

[0085] 1. Amplification system

[0086] Primers (5 groups) 10uM per group

[0087] Tris / HCl 10-100mM

[0088] Potassium chloride 1-10mM

[0089] BSA 1-5g / ml

[0090] Dithiothreitol 1-5mM

[0091] Nucleoside triphosphate and deoxynucleoside triphosphate equal concentration mixture 200-1000uM

[0092]Reverse transcriptase 5-300U

[0093] RNase H 5-300U

[0094] Phage T7 ribonucleic acid polymerase 5-300U

[0095] Negative control is RNase-free water

[0096] Positive controls are 5pM artificially synthesized avian influenza virus H5 subtype HA gene sequence, avian influenza virus H7 subtype HA gene, SARS coronavirus nucleocapsid protein gene, Hantaan virus nucleocapsid protein gene sequence, Yersinia pestis pPCP1 gene sequence, Bacillus anthracis hypothetical protein gene sequence.

[0097] 2. Detection system

[0098] Detection probes (5 groups, all labeled with digoxin) 26μM

[0099] C...

Embodiment 3

[0109] The method for detecting infectious disease pathogens that may exist in biological samples is also the method for using the kit of the present invention.

[0110] 1. Nucleic acid extraction

[0111] Take the sputum sample to be tested, centrifuge to get the supernatant, add 1ml guanidine isothiocyanate, mix well, then add 1ml TRIZOL, oscillate and mix, place in ice bath for 5 minutes, add 350ul chloroform, fully oscillate and mix, static Immediately after layering, centrifuge at 4°C, 12000r / min for 20 minutes. Transfer the supernatant to another centrifuge tube, add an equal volume of isopropanol (pre-cooled at 4°C) and mix well. Place at -20°C for 1h, then at 4°C, Centrifuge at 12000r / min for 20 minutes to precipitate total RNA. Add 0.5ml of 75% ethanol to wash, centrifuge at 12000r / min for 10 minutes at 4°C, pour off the ethanol carefully, leave at room temperature for 10 minutes, add appropriate amount of DEPC-treated water to dissolve the precipitate, The nucleic a...

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Abstract

The invention discloses a method for detecting infectious disease pathogens possibly existing in a biological sample. The infectious disease pathogens comprise chlamydia pneumoniae, haemophilus influenzae, mycoplasma pneumoniae, pneumoniae streptococcus and legionella pneumophila. The method comprises the following steps: expanding nucleic acid fragments of the biological sample, and detecting the nucleic acid fragments by using a probe. The invention also provides primers used for expanding and the probe used for detection. The invention also provides a kit comprising the primers. The method has the advantages of high sensitivity, strong specificity, simple operation and wide sample range, can simultaneously detect various infectious disease pathogens, and is suitable for early diagnosis of respiratory infectious diseases.

Description

technical field [0001] The invention relates to molecular biology, in particular to a method and a kit for detecting infectious disease pathogens. Background technique [0002] In the early stage of the epidemic of infectious diseases, accurate, fast and convenient detection of the pathogens of infectious diseases is the key to controlling the epidemic of infectious diseases. Although domestic and international surveillance systems for infectious diseases have been established, the existing detection methods have their own limitations. Serological tests detect pathogens by combining corresponding antibodies with antigens. This method is fast, simple, and easy to operate. It is suitable for early diagnosis of infectious diseases. [0003] The pathogen isolation method is to detect and identify pathogens by directly isolating and culturing pathogens. This method has high sensitivity and strong specificity, but the operation is complicated and time-consuming (it takes at lea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/21C12R1/46C12R1/01
Inventor 于常海刘乐庭冯晓燕
Owner HAI KANG LIFE
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