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Method and kit for detecting pathogens of infectious diseases

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

Inactive Publication Date: 2009-12-16
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 detect multiple infectious disease pathogens at the same time, and provide a method for detecting infectious disease pathogens that may exist in biological samples, and the infectious disease pathogens include avian influenza virus H5 subtype , H7 subtype of avian influenza virus, SARS coronavirus, Hantavirus and Yersinia pestis, including:

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Design and prepare primers and probe sequences.

[0074] According to the NCBI search, the specific gene sequences of H5 subtype of avian influenza virus, H7 subtype of avian influenza virus, SARS coronavirus, Hantavirus, Yersinia pestis and Bacillus anthracis were selected, which are respectively:

[0075] Avian influenza virus H5 subtype HA gene sequence, GENEBANK accession number is FM160642, select the highly conserved part of the sequence as the target nucleic acid sequence, the target sequence selected in the present invention is HA gene 1457-1653, its nucleotide sequence is as SEQ ID NO.1 shown;

[0076] Avian influenza virus H7 subtype HA gene sequence, GENEBANK accession number is AB302789, the target nucleic acid sequence that the present invention selects is 452-764 of HA gene, and its nucleotide sequence is as shown in SEQ ID NO.2;

[0077] The nucleocapsid protein gene sequence of SARS coronavirus, GENEBANK accession number is DQ898174, the target nucleic ...

Embodiment 2

[0090] The kits described in the present invention are prepared. Composed of:

[0091] 1. Amplification system

[0092] Primers (6 groups) each group 10uM

[0093] Tris / HCl 10-100mM

[0094] Potassium chloride 1-10mM

[0095] BSA 1-5g / ml

[0096] Dithiothreitol 1-5mM

[0097] Nucleoside triphosphate and deoxynucleoside triphosphate equal concentration mixture 200-1000uM

[0098] Reverse transcriptase 5-300U

[0099] RNase H 5-300U

[0100] Phage T7 ribonucleic acid polymerase 5-300U

[0101] Negative control is RNase-free water

[0102] Positive controls are 5pM artificially synthesized avian influenza virus H5 subtype HA gene sequence, avian influenza virus H7 type HA gene, SARS coronavirus nucleocapsid protein gene, Hantaan virus nucleocapsid protein gene sequence, Yersinia pestis pPCP1 Gene sequence, Bacillus anthracis putative protein gene sequence.

[0103] 2. Detection system

[0104] Detection probes (6 groups, all labeled with digoxin) 26μM

[0105] Captur...

Embodiment 3

[0115] 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.

[0116] 1. Nucleic acid extraction

[0117] 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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PUM

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Abstract

The invention discloses a method for detecting pathogens of infectious diseases, which possibly exist in a biological sample. The pathogens of the infectious diseases comprise avian influenza virus H5 subtype, avian influenza virus H7 subtype, SARS coronavirus, hanta virus, plague yersinia pestis and bacillus anthracis. The method comprises amplifying a nucleic acid fragment of a biological sample and detection by a probe. The invention also provides a primer for amplification and a probe for detection. The invention also provides a kit including the primer. The method has the advantages of high flexibility, strong specificity, easy operation, wide sample range, the detection for the pathogens of various infectious diseases at the same time and the suitability 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, rapid and convenient detection of 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 least ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93C12R1/01
Inventor 于常海刘乐庭冯晓燕
Owner HAI KANG LIFE
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