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Method and kit for rapidly detecting salmonella living cells in feed by combining ethidium monoazide (EMA) and polymerase chain reaction (PCR)

A Salmonella, live cell technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. The effect of positive rate

Inactive Publication Date: 2012-08-22
河南省兽药监察所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current PCR assays are prone to false-positive results, resulting in huge waste and unnecessary recalls
The reason for this result is that PCR technology, like other DNA methods, cannot distinguish between live and dead Salmonella, so this causes PCR technology to provide false results

Method used

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  • Method and kit for rapidly detecting salmonella living cells in feed by combining ethidium monoazide (EMA) and polymerase chain reaction (PCR)
  • Method and kit for rapidly detecting salmonella living cells in feed by combining ethidium monoazide (EMA) and polymerase chain reaction (PCR)
  • Method and kit for rapidly detecting salmonella living cells in feed by combining ethidium monoazide (EMA) and polymerase chain reaction (PCR)

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specific Embodiment approach

[0028] The present invention will be further described below in conjunction with embodiment:

[0029] Such as figure 1 As shown, the casing 1 of the covalent cross-linking exposure device of the present invention is provided with a visible window 3, a dodge door 5, a halogen lamp switch 6, an ultraviolet lamp switch 7 and a main power switch 8, and the dodge door 5 and the casing 1 hinged, a 500W halogen lamp 4 is provided on the inner wall of the top of the box body 1, and two ultraviolet lamp tubes 2 are respectively installed on the inner walls of both sides of the box body 1, and the two parallel ultraviolet lamp tubes 2 are arranged symmetrically on the left and right sides of the box body The inner side of body 1. The halogen lamp 4 provides strong light, and the ultraviolet lamp tube 2 is used for sterilization. In order to improve the sterilization effect, two ultraviolet lamp tubes 2 are respectively installed on the inner walls of both sides of the box body 1 . Ob...

Embodiment 1

[0032] The kit of the present invention includes a Salmonella positive control, a Salmonella negative control, Taq premixed enzyme and primer mixture of appropriate concentration, DL Marker 2000, EMA solution of appropriate concentration and a covalent cross-linking exposure device. The premixed enzyme is 20u / mL, the primer concentration is 100pmol / mL, and the storage condition is -20°C. The sequences of the primers are: SE-1: GTGAAATTATCGCCACGTTCGGG SE-2: CATCGCACCGTCAAAGGAAC. EMA solution, its concentration is 0.5mg / mL.

[0033] Method of the present invention comprises the steps:

[0034] 1) Enrich the feed sample with BPW broth, pipette 500ul of bacterial suspension into four 1.5mL centrifuge tubes respectively, two of which are prepared as live cell bacterial suspension, and prepare two of them in a 100°C water bath for 10 min into a dead cell suspension. Take one part of the live cell suspension and one part of the dead cell suspension, add 4ul 0.5mg / mL hexidine azide...

Embodiment 2

[0039] The kit includes a Salmonella positive control, a Salmonella negative control, Taq premixed enzyme and primer mixture at an appropriate concentration, DL Marker 2000, an EMA solution at an appropriate concentration, and a covalent cross-linking exposure device. The premixed enzyme is 20 u / mL, the primer concentration is 100 pmol / mL, and the storage condition is -20°C. The sequences of the primers are: SE-1: GTGAAATTATCGCCACGTTCGGG SE-2: CATCGCACCGTCAAAGGAAC. EMA solution, its concentration is 0.5mg / mL.

[0040] Detection method of the present invention comprises the following steps:

[0041] 1) Take 2 parts of the sterilized blank concentrated feed and compound feed, add 1 CFU of Salmonella per 25 grams, enrich the bacteria in the sterilized BPW broth, draw 500ul of the bacterial suspension into four 1.5mL centrifuge tubes, Add 4ul 0.5mg / mL hexidine azide bromide solution to two of the centrifuge tubes respectively, place the centrifuge tubes on the ice box, and expose ...

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Abstract

The invention discloses a method and a kit for rapidly detecting salmonella living cells in feed by combining ethidium monoazide (EMA) and polymerase chain reaction (PCR). The kit comprises salmonella positive control, salmonella negative control, a Taq premix enzyme and a primer mixture, DL Marker2000, an EMA solution and a covalent cross-linking exposure device. The method is low in cost and convenient and fast in operation, and can objectively reflect the existence condition of the salmonella living cells in the feed, thus providing an effective means for rapid and accurate detection of the salmonella in the feed.

Description

technical field [0001] The invention relates to a method and a kit for detecting Salmonella living cells in feed, in particular to a method and a kit for rapidly detecting Salmonella living cells in feed combined with EMA and PCR. Background technique [0002] Animal feed is an important link in the "from farm to table" food safety chain. Eating animals or animal products contaminated by pathogenic bacteria may cause human diseases. Therefore, accurate and rapid detection of Salmonella in feed is important for preventing contamination of livestock, poultry and humans with Salmonella is of great significance. The traditional Salmonella detection method is far from meeting the requirements of modern detection due to its shortcomings such as long detection cycle, high missed detection rate, complicated procedures, and various reagents required. Real-time PCR technology has been used for many years to detect pathogenic microorganisms in food or feed. The traditional PCR detect...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10
CPCY02A50/30
Inventor 班付国高延玲张发旺刘宏伟周红霞黄京燕马俊韩立高小玲焦玉萍朱松波宋志超舒畅李华岑李金磊董鹏陈晓鸽贾振民
Owner 河南省兽药监察所
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