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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 effect of fast detection, high efficiency and short time

Active Publication Date: 2014-07-16
海康生物科技(北京)有限公司
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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. The infectious disease pathogens include Chlamydia psittaci, Karl Pneumocystis, Leptospira, Rickettsia Q fever, and Brucella, 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 psittaci, Pneumocystis carinii, Leptospira, Q-fever Rickettsia and Brucella were selected, which are respectively:

[0070] Chlamydia psittaci endosomal membrane protein A gene sequence, GENEBANK accession number is DQ117471, select the highly conserved part of the sequence as the target nucleic acid sequence, the target sequence selected in the present invention is membrane protein A gene 317-472, its nucleotide sequence is as SEQ ID As shown in NO.1;

[0071] The main surface glycoprotein gene sequence of Pneumocystis carinii, the GENEBANK accession number is AF033209, the target nucleic acid sequence selected in the present invention is the main surface glycoprotein gene 2895-3010, and its nucleotide sequence is shown in SEQ ID NO.2;

[0072] Leptospira gyrase B subunit gene sequence, GENEBANK accession number is AY896758, the target nuclei...

Embodiment 2

[0083] The kits described in the present invention are prepared. Composed of:

[0084] 1. Amplification system

[0085] Primers (5 groups) 10uM per group

[0086] Tris / HCl 10-100mM

[0087] Potassium chloride 1-10mM

[0088] BSA 1-5g / ml

[0089] Dithiothreitol 1-5mM

[0090] Nucleoside triphosphate and deoxynucleoside triphosphate equal concentration mixture 200-1000uM

[0091] Reverse transcriptase 5-300U

[0092] RNase H 5-300U

[0093] Phage T7 ribonucleic acid polymerase 5-300U

[0094] Negative control is RNase-free water

[0095] Positive controls are 5pM artificially synthesized Chlamydia psittaci endosome membrane protein A gene sequence, Pneumocystis carinii major surface glycoprotein gene sequence, Leptospira gyrase B subunit gene sequence, Q heat rickettsia outer membrane Protein gene sequence, Brucella outer membrane protein omp2b gene sequence.

[0096] 2. Detection system

[0097] Detection probes (5 groups, all labeled with digoxin) 26μM

[0098] Capt...

Embodiment 3

[0108] 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.

[0109] 1. Nucleic acid extraction

[0110] Take the nasopharyngeal secretion sample to be tested, centrifuge to get the supernatant, add 1ml guanidine isothiocyanate, mix well, then add 1ml TRIZOL, shake and mix, place in ice bath for 5 minutes, add 350ul chloroform, shake and mix well, After static layering, immediately 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, 4 Centrifuge at 12000r / min for 20 minutes at 12000r / min at ℃, and precipitate total RNA. Add 0.5ml of 75% ethanol to wash, centrifuge at 12000r / min at 4℃ for 10 minutes, pour off the ethanol carefully, leave at room temperature for 10 minutes, add appropriate amount of DEPC-treated water to dissolve Precipitate ...

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PUM

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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 psittaci, pneumocystis carinii, leptospira, Q fever rickettsiosis and brucellosis. 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): C12Q1/68C12N15/11C12R1/01C12R1/90
Inventor 于常海刘乐庭冯晓燕
Owner 海康生物科技(北京)有限公司
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