Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Salmonella constant-temperature fluorescent detection primer combination capable of avoiding false negative, reagent kit and detection method

A Salmonella and fluorescence detection technology, which is applied in the field of molecular biology detection, can solve the problems of complex detection samples and the hazards of Salmonella, and achieve the effect of simple identification, high specificity and high specificity

Inactive Publication Date: 2016-01-06
GUANGDONG NO 2 PROVINCIAL PEOPLES HOSPITAL +1
View PDF6 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the test samples are complicated. Because the current detection methods do not adopt monitoring methods for false negative test results, there is a possibility of missed detection. However, Salmonella is a serious hazard, and the damage caused by missed detection is immeasurable. In addition, PCR amplification requires expensive instruments. Many grassroots testing units do not have the corresponding testing conditions

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Salmonella constant-temperature fluorescent detection primer combination capable of avoiding false negative, reagent kit and detection method
  • Salmonella constant-temperature fluorescent detection primer combination capable of avoiding false negative, reagent kit and detection method
  • Salmonella constant-temperature fluorescent detection primer combination capable of avoiding false negative, reagent kit and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: Establishment of Salmonella Constant Temperature Fluorescence Detection Kit

[0064] Constant temperature fluorescent detection kit for Salmonella, including detection primer set, internal standard primer set, reaction solution, Bst DNA polymerase, positive control, negative control and internal standard.

[0065] (1) Detection primer set: The Salmonella SPI gene was used as the target gene to design primers. The detection primer set included a pair of outer primers, a pair of inner primers and a pair of loop primers, and their nucleotide sequences were as follows:

[0066] SPI-F3: 5'-TTTACGGTTCCTTTGACGG-3';

[0067] SPI-B3: 5'-GCCCCATATTATCCGTATCG-3';

[0068] SPI-FIP: 5'-AGTAGACAGGGCGGAGGACTATTGGCGGTATTTCGGTG-3';

[0069] SPI-BIP: 5'-ACCATGCTGACCATTGGTGATGCGGCACTAATCGCAATCA-3';

[0070] SPI-LF: 5'-AATCCATACCATGGCGAGTC-3';

[0071] SPI-LB: 5'-GTCTTGTCGCCCAGATCC-3'.

[0072] (2) Internal standard primer set: use the Salmonella SPF gene as the internal ...

Embodiment 2

[0083] Embodiment 2: The constant temperature fluorescence detection method of Salmonella

[0084] Utilize the constant temperature fluorescence detection kit of the Salmonella of embodiment 1 to detect the sample, the steps are as follows:

[0085] (1) Extract the DNA of the sample to be tested.

[0086] (2) Using the primer composition of claim 1 to carry out constant temperature amplification of the DNA of the sample to be tested:

[0087] The 25μl reaction system for constant temperature amplification contains: SPI-F30.2μM, SPI-B30.2μM, SPI-FIP1.6μM, SPI-BIP1.6μM, SPI-LF0.8μM, SPI-LB0.8μM, SPF-F30.2μM , SPF-B30.2μM, SPF-FIP1.6μM, SPF-BIP1.6μM, SPF-LoopF0.8μM, SPF-LoopB0.8μM, reaction solution 12.5μl, DNA polymerase 8U, 10×SYBRGreenI0.5μl, sample to be tested 2μl, internal standard 2μl, make up to 25μl with ultrapure water;

[0088] The program of constant temperature amplification is: react at 63-65°C for 30-45min, and continue at 80°C for 2min.

[0089] (3) Judgment o...

Embodiment 3

[0094] Embodiment 3: Sensitivity experiment

[0095] The constructed plasmid was subjected to a sensitivity test, and the 1pg / μl plasmid was diluted 10 times into four gradients of 1pg / μl, 100fg / μl, 10fg / μl, and 1fg / μl as quality control standards, and the method in Example 2 was used for detection , determine the minimum detection limit of the internal standard gene by sensitivity experiment 10fg / μl (such as figure 1 shown), and review the minimum detection limit ( figure 2 ), and set the internal standard concentration as the concentration of the lowest detection limit.

[0096] Cultivate the cultured Salmonella, perform 10-fold serial dilution of the cultured bacteria, extract DNA, and count the diluted bacterial solution on a plate, and compare the plate culture counting results with the lowest sensitivity of this kit. The minimum detection degree is 1.6×10 3 CFU / mL, test results see image 3 .

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a salmonella constant-temperature fluorescent detection primer combination capable of avoiding false negative, a reagent kit and a detection method. The salmonella constant-temperature fluorescent detection primer comprises a detection primer group and an internal standard primer group, wherein the detection primer group is designed according to a salmonella SPI gene, and a sequence of the detection primer group is as shown in SEQ ID NO.1 to 6; the internal standard primer group is designed according to a salmonella SPF gene, and a sequence of the internal standard group is as shown in SEQ ID NO.7 to 12. Besides the primer groups, the salmonella constant-temperature detection reagent kit also comprises a DNA polymerase, reaction liquid, an internal standard target fragment, a positive reference and a negative reference. The detection primer, the reagent kit and the detection method have advantages of rapidness, high efficiency, simplicity in operation, high specificity, high sensitivity, suitability for the on-site detection and the like, can effectively prevent the occurrence of false negative and are suitable for being popularized to use.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and in particular relates to a constant temperature detection primer set, kit and detection method for Salmonella. Background technique [0002] salmonella( salmonella ) is a common zoonotic pathogen, which not only causes various animal diseases, but also is related to many human diseases. Among them, the food poisoning cases caused by Salmonella are in the forefront of food poisoning. Members of the Salmonella genus can infect a variety of animals and humans, most of which are highly pathogenic. Salmonella infection has been widely paid attention to because of its harm to human beings and livestock and poultry breeding industry. [0003] At present, many countries including my country generally adopt the traditional culture method for the detection of Salmonella, and it takes about 4 to 5 days to report the test results. In addition, the detection method is cumbersome, tim...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/42
CPCC12Q1/689C12Q2600/166
Inventor 曹东林胡亮杉林茂锐石磊周旋钟丽梅方晓琳陈洵唐大运张璜申洪杰雷达张知洪田军章
Owner GUANGDONG NO 2 PROVINCIAL PEOPLES HOSPITAL
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com