Salmonella molecular detection kit based on bcfD genes and non-diagnostic detection method

A Salmonella and molecular detection technology, applied in the field of molecular biology detection, can solve the problems of rapid batch diagnosis of Salmonella and complex operation, etc., and achieve the effect of being suitable for large-scale promotion and application, convenient operation, and broad market prospects

Active Publication Date: 2014-07-02
SINOPHARM YANGZHOU VAC BIOLOGICAL ENG CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] With the continuous development of molecular biology, traditional bacteriological detection methods can no longer meet the rapid batch diagnosis of Salmonella. The identification of pathogenic microorganisms is no longer limited to morphological inspections, and has developed to detect pathogenic nucleic acids and pathogens at the molecular level. For biological macromolecules such as pro

Method used

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  • Salmonella molecular detection kit based on bcfD genes and non-diagnostic detection method
  • Salmonella molecular detection kit based on bcfD genes and non-diagnostic detection method
  • Salmonella molecular detection kit based on bcfD genes and non-diagnostic detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 is based on the design of the LAMP detection primer of Salmonella bcfD pilus gene:

[0048] The standard strain of Salmonella ATCC14028 preserved in the laboratory was sequenced after nucleic acid amplification, and homology analysis was performed by BLAST software to obtain the broad-spectrum and specific conserved target sequence bcfD gene of Salmonella (sequence has been uploaded to the American Gene Database; GenBank Accession number: KJ021967, as shown in SEQ ID NO.7), and then according to the conserved target gene sequence, use the online design software: Primer Explorer version4 ( http: / / primerexplorer.jp / e ) to design the LAMP primer set. As a result, three sets of primer pairs were optimized, F3 and B3 as the first set, FIP and BIP as the second set, and LF and LB as the third set. The specific primer sequences are shown in Table 1. In the application, the three sets of primer pairs can be mixed and used in any proportion, and a specific optimize...

Embodiment 2

[0053] Example 2 Establishment of the detection method of the molecular detection kit based on the Salmonella bcfD pili gene:

[0054] Use the three pairs of primers obtained in Example 1 for carrying out LAMP detection to Salmonella to carry out LAMP detection to various types of Salmonella preserved in the laboratory to obtain the best reaction system and reaction conditions. The specific methods are as follows:

[0055] (1) Sample collection: Salmonella molecular detection kit based on bcfD gene, the samples include 15 Salmonella standard strains and 10 non-Salmonella standard strains used in the specificity verification experiment and the pigs, cattle, Strains isolated from the cloaca of chickens, ducks, and geese and their tissues, as well as strains isolated from live poultry animals and animal-derived products such as eggs and milk sold on the market.

[0056] (2) Extraction of genomic DNA of the sample to be tested: extraction according to conventional methods or kits....

Embodiment 3

[0081] Embodiment 3 Sensitivity evaluation experiment

[0082] This embodiment tests the sensitivity of the LAMP detection method of the present invention and the common PCR method for detecting Salmonella. The method is: first culture the clinical Salmonella and other negative strains (refer to GB4789.4-2010), and culture the bacteria (the original concentration is 5.6×10 7 cfu / μL) for 10-fold serial dilution, extract DNA according to the method in Example 2, and perform LAMP and PCR detection respectively, wherein the PCR primers are the outer primers (F3 and B3) of the LAMP method, and the diluted bacterial solution is plated at the same time For culture counting, compare the plate culture counting results with the lowest sensitivity of LAMP in this kit. Wherein: the LAMP detection reaction system is shown in Table 2.

[0083] Table 2 LAMP detection reaction system

[0084]

[0085] The above reaction system was prepared in a PCR tube, and a positive control and a neg...

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Abstract

The invention discloses a salmonella molecular detection kit based on bcfD genes and a non-diagnostic detection method. The kit comprises (1) 10* Lamp buffer; (2) 8000U/mL BstDNA polymerase; (3) 10mM dNTPs; (4) a Lamp primer group: outer primer 5pmol/micro liter, inner primer 40pmol/micro liter, and annular primer 20pmol/micro-liter; (5) double distilled water; (6) fluorescent dye: 10*SYBR Green I; (7) positive control: salmonella standard strain genome DNA; and (8) negative control: double distilled water. The LAMP detection method has the advantages of rapidness, high efficiency, simplicity in operation, high specificity, high sensitivity, broad-spectrum property, applicability to in-situ detection and the like; no complicated instrument is needed; novel technical support is provided for the detection of salmonella; the salmonella molecular detection kit can be used for screening and detecting salmonella in animal husbandry production units, basic medical sanitary units, entry-exit and different disease control centers and is wide in market prospect, good in economic benefit and social benefit and wide in application range.

Description

technical field [0001] The invention relates to the technical field of molecular biology detection, in particular to a bcfD gene-based Salmonella molecular detection kit and a non-diagnostic detection method thereof. Background technique [0002] Salmonella (Salmonella) is the most common zoonotic pathogen in Enterobacteriaceae, often parasitic in the intestinal tract of animals and humans. Salmonella is an important foodborne bacterium that causes human infection in both developed and developing countries. Food poisoning caused by Salmonella is the highest proportion of all bacterial food poisoning and the most harmful one, accounting for about 42.6%-60% of bacterial food poisoning. At present, the detection of Salmonella in China is still dominated by conventional bacteriological detection methods, and its shortcomings are mainly manifested in: First, the operation steps are cumbersome and time-consuming. The detection of Salmonella in the current national or industry sta...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/42
CPCC12Q1/6844C12Q2531/119C12Q2563/107
Inventor 龚建森许明贾雪波庄林林王成明朱春红刘学贤徐步窦新红童海兵邹剑敏
Owner SINOPHARM YANGZHOU VAC BIOLOGICAL ENG CO LTD
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