RPA primer composition and method for detecting shiga toxin-producing Escherichia coli

An Escherichia coli and Shiga toxin-producing technology, applied in the biological field, can solve the problems of lack of degenerate bases, unclear genotype range, lack of detectable genotypes in test data, etc. Simple to use effects

Inactive Publication Date: 2020-08-07
SHANGHAI INST FOR FOOD & DRUG CONTROL
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although, some researchers have developed RPA methods for detecting STEC (MURINDA SE, IBEKWE AM, ZULKAFFLY S, et al., Real-Time Isothermal Detection of Shiga Toxin–Producing Escherichia coli Using Recombinase Polymerase Amplification[J]. Foodborne Pathogens&Disease, 11( 7):529-536), but compared its...

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
  • RPA primer composition and method for detecting shiga toxin-producing Escherichia coli
  • RPA primer composition and method for detecting shiga toxin-producing Escherichia coli
  • RPA primer composition and method for detecting shiga toxin-producing Escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] This embodiment provides an RPA primer composition and probe for detecting Shiga toxin-producing Escherichia coli. The specific design process includes the following steps:

[0072] Download stx from the GeneBank database (www.ncbi.nlm.nih.gov / genbank)1 and stx 2 Nucleic acid sequence of genotype subtype, including stx 1 Three genotypes (stx 1a , stx 1c , stx 1d ) and stx 2 7 genotypes (stx 2a , stx 2b , stx 2c , stx 2d , stx 2e , stx 2f , stx 2g ). Primer Premier 5.0 software was used to design primers and probe combinations (Table 1) according to the RPA design principles of TwistDx Company, and were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0073] Table 1 RPA primer and probe sequences for detection of Shiga toxin-producing Escherichia coli

[0074]

[0075] Use RPA technology to screen suitable RPA amplification primers. The specific operation steps are as follows:

[0076] (1) will detect stx 1 and stx 2 The ups...

Embodiment 2

[0082] This embodiment provides a method for detecting Shiga toxin-producing Escherichia coli, using fluorescent RPA technology; including the following steps:

[0083] In step 1, fluorescent RPA reaction uses fluorescent DNA constant temperature rapid amplification kit (Anpu Future Biotechnology Co., Ltd.). Add 2.95 μL of reaction premix A to the dry powder reaction tube, 0.1 μL each of the primers screened in Example 1 and the probe (10 μmol / L) provided, 1.5 μL of DNA template, and finally add buffer B (containing 280 mmol / L Mg 2+ ) 0.25 μL to make a 5 μL reaction system.

[0084] Step 2: Mix well, set at 39°C (temperature range: 37-42°C) for constant temperature reaction for 20min, and collect fluorescence signal every 30s for a total of 40 cycles.

[0085] Step 3: After the reaction is completed, the data is read and analyzed.

[0086] By detecting genomic nucleic acid and bacterial cells, the sensitivity of the method using fluorescent RPA technology provided by this em...

Embodiment 3

[0092] This example provides a method for detecting Shiga toxin-producing Escherichia coli, using fluorescent RPA microfluidic chip technology to simultaneously detect 9 gene subtypes of stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e and stx2g .

[0093] Among them, the disk centrifugal microfluidic chip assembly and chip detector (SX-MA2000 plus) used in the experiment were provided by Shanghai Suxin Biotechnology Co., Ltd. The chip is made of polycarbonate material and micro-injected into a disc structure. Each chip has 8 sets of identical microfluidic structure areas, and each area has 1 sample loading area, 1 liquid distribution area and 4 independent reactions. area, 32 reactions can be carried out simultaneously.

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

PropertyMeasurementUnit
Sensitivityaaaaaaaaaa
Sensitivityaaaaaaaaaa
Sensitivityaaaaaaaaaa
Login to view more

Abstract

The invention discloses an RPA (recombinase polymerase amplification) primer composition and method for detecting shigella toxin-producing Escherichia coli. The primer composition is used for detecting nine gene subtypes, namely, stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e and stx2g and comprises a primer pair with sequences shown as SEQ ID NO: 1 and SEQ ID NO: 4 and a primer pair withsequences shown as SEQ ID NO: 9 and SEQ ID NO: 12. The method provided by the invention has the advantages of simple operation, high specificity and high sensitivity, is suitable for on-site rapid detection, can complete nucleic acid detection within 20 min, and can provide a rapid screening result for supervision and inspection of listed products and emergency treatment of supervision departments. Meanwhile, the primer and probe combination designed and screened for the first time can effectively detect nine stx gene subtypes, namely stx1a, stx1c, stx1d, stx2a, stx2b, stx2c, stx2d, stx2e andstx2g genes. And in combination with a centrifugal disc type micro-fluidic chip technology, rapid and high-throughput detection on STEC in foods, medicines and livestock breeding products can be realized within an extremely small volume (5 microliters ).

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an RPA primer composition and method for detecting Shiga toxin-producing Escherichia coli. Background technique [0002] Shiga toxin-producing Escherichia coli (STEC) is a class of food-borne pathogenic microorganisms that can cause diarrhea, intestinal bleeding, hemolytic uremic syndrome, renal failure, hemolytic anemia and death in humans, and can exist in water bodies, plants , animals, especially ruminants. STEC bacteria below 100CFU in food may cause human disease. There are more than 400 serotypes of STEC. According to data released by the US Centers for Disease Control and Prevention, there were 5,441 confirmed cases of STEC infection in the US in 2016 alone, with an estimated number of more than 260,000 patients and 30 deaths. [0003] Currently, the main molecular target for detecting STEC is the stx gene encoding Shiga toxin. Both the International Organization for Stand...

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): C12Q1/689C12Q1/6844C12Q1/10C12R1/19
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2531/119C12Q2522/101
Inventor 范一灵王淑娟李琼琼宋明辉秦峰刘浩杨美成
Owner SHANGHAI INST FOR FOOD & DRUG CONTROL
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap