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Kit for detecting streptococcus suis type 2 and application of kit

A technology of Streptococcus suis and a kit, which is applied in the direction of microorganism-based methods, microorganism measurement/inspection, microorganisms, etc., can solve the problems of complicated operation and long detection time, and achieve high sensitivity, rapid and accurate detection, and strong specificity Effect

Active Publication Date: 2013-11-27
长沙市疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] CN102605046A discloses a kit and a method for detecting Streptococcus suis type 2. The kit includes an upstream primer, a downstream primer and a probe, and the 5' end of the upstream primer and / or the 5' end of the downstream primer are covered by biotin modification; the 5' end and / or 3' end of the probe is modified by digoxin; the probe can hybridize with the biotin-modified single-stranded DNA in the double-stranded DNA amplification product, This method has strong specificity, but the method is complicated to operate, takes a long time to detect, and its sensitivity has a lot of room for improvement

Method used

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  • Kit for detecting streptococcus suis type 2 and application of kit
  • Kit for detecting streptococcus suis type 2 and application of kit
  • Kit for detecting streptococcus suis type 2 and application of kit

Examples

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Embodiment 1

[0029] Embodiment 1: kit of the present invention and the method for detecting Streptococcus suis type 2

[0030] Make the LAMP-based S. suis2 capsular polysaccharide CPS2J gene detection kit according to the following formula:

[0031] (1) Amplification reaction solution: 20.0 μL of each tube of amplification reaction solution consists of: 10×Bst DNA Polymerase Buffer 2.5 μl, dNTP Mixture (10mM) 3.5 μl, Betaine (5M) 5.0 μl, Mgso 4 (50mM) 1.6μl, 7.4μL ddH 2 o.

[0032] (2) Mixed primer solution: including 10 uM each of CPS2J-F3 and CPS2J-B3 primers; 40 uM each of CPS2J-FIP and CPS2J-BIP primers.

[0033] (3) Bst DNA Polymerase: 8U / μl;

[0034] (4) S.suis2 type positive standard product is S.suis2 type strain genomic DNA (1~100nM);

[0035] (5) The negative control substance is ddH 2 o.

[0036] The above kit of the present invention is used to detect the S. suis2 type CPS2J gene according to the following procedures:

[0037] (1) Bacterial DNA extraction: the bacterial ...

Embodiment 2

[0045] Embodiment 2: the accuracy measurement of kit of the present invention and detection method

[0046] Get certain samples 1, 2, and 3 to be tested, and use the detection method described in Example 1 of the present invention to carry out positive detection of Streptococcus suis type 2, and the results show that sample 2 to be checked contains Streptococcus suis type 2, and samples 1 and 3 to be tested Does not contain Streptococcus suis type 2. The gene sequencing and identification of the LAMP amplification product of the sample to be tested 2 was carried out, and the sequencing results showed that the sequence of the LAMP amplification product of the sample to be tested 2 was 100% homologous to the corresponding S. suis2 type-specific capsular polysaccharide CPS2J gene sequence , which fully demonstrates the accuracy of the present invention, and the kit and detection method of the present invention can identify the S. suis2 type CPS2J gene.

Embodiment 3

[0047] Embodiment 3: the specificity analysis of kit of the present invention and detection method

[0048] Staphylococcus aureus (CMCC-26003-25), Staphylococcus aureus enterotoxigenic SEA bacteria (ATCC-13565), Staphylococcus aureus enterotoxigenic SEB bacteria (ATCC-14458), Staphylococcus aureus enterotoxigenic bacteria SEC bacteria (ATCC-19095), Staphylococcus aureus enterotoxigenic SEE bacteria (ATCC-27664), Salmonella enteritidis (ATCC-13076), Salmonella typhimurium (CMCC-50115), Salmonella typhi (CMCC-50071) , Shigella dysenteriae (CMCC-51252), Escherichia coli O157: H7 (CMCC-44050-3), Vibrio parahaemolyticus (VPL4-90), Proteus mirabilis (CMCC-49005), Shigella flexneri ( CMCC-51572), Enterotoxigenic Escherichia coli (CMCC-44824-3), Enteroinvasive Escherichia coli (CMCC-44825-3), Enteropathogenic Escherichia coli (CMCC-44155-10) , enterohemorrhagic Escherichia coli (CMCC-44050-3) is a control bacterium, utilizes the kit described in the embodiment of the present inventio...

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Abstract

The invention relates to a kit for detecting streptococcus suis type 2 nucleic acid by an isothermal nucleic acid amplification method and application of the kit to detection of streptococcus suis type 2 nucleic acid. The kit comprises amplification reaction liquid, primer reaction liquid, DNA (deoxyribonucleic acid) polymerase, nucleic acid dye, an S.suis type 2 positive standard product and a negative control product. A method for applying the kit to S.suis type 2 positive detection comprises five steps of extracting DNA of to-be-tested specimen bacteria, performing loop-mediated isothermal amplification (LAMP) on S.suis type 2 CPS2J gene, performing electrophoretic detection on an amplification product, performing chromogenic reaction of the LAMP amplification product and judging positive of S.suis type 2 streptococcus. The kit has the advantages of quickness, convenience, strong specificity and high sensitivity when being applied to positive detection of the S.suis type 2 streptococcus, and is very suitable for quick detection of the S.suis type 2 streptococcus in medical or food industry.

Description

technical field [0001] The invention relates to a kit for detecting pathogenic microorganisms and its application, in particular to a kit for detecting Streptococcus suis type 2 nucleic acid by an isothermal nucleic acid amplification method and its application in the detection of Streptococcus suis type 2 nucleic acid, belonging to The field of detection of pathogenic microorganisms. Background technique [0002] Streptococcus suis (S. suis) type 2 is an important zoonotic pathogen with strong pathogenicity, which can cause infection and death in pigs and humans. In 1998, the outbreak of S.suis2 in pigs occurred in Nantong City, Jiangsu Province, and the first case of human infection with S.suis2 was reported in China. The epidemic has become an important public health problem. [0003] Existing detection methods for Streptococcus suis include microbiological methods, serological identification, and PCR detection. The microbiological detection method is based on the bact...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12R1/46
Inventor 张如胜陈法明孙边成欧新华
Owner 长沙市疾病预防控制中心
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