Nucleic acid isothermal amplification detection kit for Salmonella and detection method

A technology for constant temperature amplification detection and Salmonella nucleic acid, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve the problems of cumbersome ELISA operation, expensive reagent costs, and difficulty in popularization, and achieve Highly reproducible, fast-response, high-sensitivity results

Inactive Publication Date: 2016-02-17
ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ELISA operation is cumbersome and time-consuming; PCR detection method is fast, sensitive, and highly accurate, but requires certain equipment and technical requirements, and the cost of reagents is relatively expensive, making it difficult to popularize

Method used

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  • Nucleic acid isothermal amplification detection kit for Salmonella and detection method
  • Nucleic acid isothermal amplification detection kit for Salmonella and detection method
  • Nucleic acid isothermal amplification detection kit for Salmonella and detection method

Examples

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Comparison scheme
Effect test

Embodiment 1

[0042] Composition and preparation of embodiment 1 kit of the present invention

[0043] a) DNA extraction reagent (preferably the BacterialGenomicDNAExtractionKit of Takara Company (product number DV810A);

[0044] b) Salmonella nucleic acid constant temperature amplification reaction solution: two peripheral primers (10pmol), two probes (20pmol) and two cross primers (40pmol), 1×Thermolbuffer, MgSO 4 (6mmol), dNTPs solution (0.4mmol each), BstDNA polymerase (8U) and sterile double distilled water, the total reaction volume is 25μl;

[0045] The forward peripheral primer is: 5′-AACAGTGCTCGTTTACGACC-3′;

[0046] The reverse peripheral primer is: 5′-CTGATCGATAATGCCAGACG-3′;

[0047] Forward 5′-end Biotin labeled probe: 5′-Biotin-AGGTAGGTAATGGAATGACGA-3;

[0048] Reverse 5′ end Fitc labeled probe: 5′-Fitc-CGTTTCTACATTGACAGAATCCTCAG-3′;

[0049] Amplify the forward primer:

[0050] 5'-GCGATCAGGAAATCAACCAGATAGAATGGTGATGATCATTTCTATGTTC-3';

[0051] Amplification reverse prime...

Embodiment 2

[0059] Embodiment 2 detects the concrete method of Salmonella nucleic acid with kit of the present invention

[0060] a) Using the DNA extraction solution in the Salmonella constant temperature amplification detection kit to extract DNA from the specimen to be tested.

[0061] b) Take the sample DNA as a template and add it to a PCR tube containing a constant temperature amplification reaction solution for Salmonella, including 5 μl of sample DNA, 20 μl of reaction solution, and amplify at 65°C for 35 minutes; add positive templates to the positive and negative control PCR tubes respectively (Salmonella invA gene, SEQ ID NO.1) and negative template (double distilled water).

[0062] c) Put the reacted PCR tube into the nucleic acid test strip anti-pollution detection device (No. 3 device of Hangzhou Ustar Biotechnology Co., Ltd.) for detection, and interpret the result after 2 minutes. When the sample contains Salmonella nucleic acid, the detection line of the test strip is p...

Embodiment 3

[0064] Embodiment 3 detects the specificity of Salmonella with kit of the present invention

[0065] Detect Salmonella typhi CMCC50071, Salmonella typhimurium CMCC50115, Shigella flexneri CMCC51572, Pseudomonas aeruginosa (Pseudomonas aeruginosa) ATCC27853, Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, gutter according to the method of Example 2 Enterobacter ATCC13047, Vibrio parahaemolyticus ATCC17802, Campylobacter jejuni ATCC33291, Listeria monocytogenes ATCC19111, Clostridium difficile ATCC9689 and Salmonella detection kit positive control substance, the result shows that detection of Salmonella nucleic acid with kit of the present invention has strong Except for Salmonella typhi CMCC50071, Salmonella typhimurium CMCC50115 and the positive control of the kit, all other strains were negative. like figure 2 As shown, 1-13 in the figure respectively represent Salmonella typhi CMCC50071, Salmonella typhimurium CMCC50115, Shigella flexneri CMCC51572, Pseudomona...

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Abstract

The invention discloses a nucleic acid isothermal amplification detection kit for Salmonella and a detection method. The detection kit comprises a DNA extraction reagent, an isothermal amplification reaction liquid, a positive control and a negative control. The detection kit is high in specificity and high in sensitivity; the temperature of all nucleic acid isothermal amplification reactions is consistent, only 35 minutes are required for isothermal amplification, 1-2 minutes are required for a detection result of a nucleic acid test strip, only about 1 hour is required for the whole detection process from sample receiving to result acquisition, only one isothermal instrument is required in the whole reaction process, and the detection kit is particularly suitable for on-site and rapid detection and elimination of the Salmonella in food.

Description

(1) Technical field [0001] The invention relates to a rapid detection technology for constant temperature amplification of Salmonella nucleic acid, which is suitable for qualitative detection of Salmonella. (2) Background technology [0002] Salmonella widely exists in nature, and more than 2,500 serotypes have been discovered so far, and 216 serotypes have been found in my country. The serotypes related to human diseases are mainly concentrated in groups A to E, including: Salmonella typhi, Salmonella paratyphi A, B, C, Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella duck, Salmonella newport, etc., among which Salmonella typhimurium, Salmonella enteritidis, and Salmonella choleraesuis are the most common. Food poisoning caused by Salmonella ranks first in Japan, Europe and the United States, and it is also a common bacteria in bacterial food poisoning in my country. In my country's inland areas, 70% to 80% of bacterial food poisoning is...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/42
CPCC12Q1/6844C12Q2531/119C12Q2537/1376C12Q2565/625
Inventor 徐昌平余蓓蓓卢亦愚冯燕须周恒胡宗悦
Owner ZHEJIANG CENT FOR DISEASE CONTROL & PREVENTION
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