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Salmonella CRISPR (clustered regularlay interspaced short palindromic repeats) sequencing typing method

A technology of Salmonella and typing methods, applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, which can solve problems such as high requirements for professional equipment and software, high technical difficulty, and limited resolution. Achieve the effect of high consistency, high resolution and easy operation

Active Publication Date: 2015-07-15
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above typing methods have advantages and disadvantages. The RFLP method has good repeatability and is easy to operate, but the resolution is limited; the AFLP method has high resolution and good repeatability, but the technical difficulty is also very high; RAPD-PCR and Rep-PCR distinguish High rate, but poor repeatability; PFGE technology has high resolution and good repeatability, and is known as the "gold standard" for molecular typing of pathogenic microorganisms, but it has high requirements for professional equipment and software, and is not suitable for grassroots disease control units and facilities. Simple laboratory development, and the analysis of electrophoretic images has a certain degree of subjectivity, coupled with the inevitable manual map adjustment, which also makes this method not well suited for high-throughput analysis interpretation and data exchange

Method used

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  • Salmonella CRISPR (clustered regularlay interspaced short palindromic repeats) sequencing typing method
  • Salmonella CRISPR (clustered regularlay interspaced short palindromic repeats) sequencing typing method
  • Salmonella CRISPR (clustered regularlay interspaced short palindromic repeats) sequencing typing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Molecular typing and serotype prediction of 82 strains of Salmonella

[0034] 1. Strains:

[0035] The strains selected for this test are 82 strains of Salmonella from various regions in my country preserved by our center, which have been identified as Salmonella by traditional serology and belong to 21 serotypes (the results of traditional serological typing of the 82 strains are shown in Table 2 ).

[0036] 2. Primer synthesis: select two pairs of primers A1, A2 and B1, B2 with the widest amplification range in the primer table

[0037] A1 (5′-GTRGTRCGGATAATGCTGCC-3′)

[0038] A2 (5'-CGTATTCCGGTAGATBTDGATGG-3')

[0039] B1 (5′-GAGCAATACYYTRATCGTTAACGCC-3′)

[0040] B2 (5′-GTTGCDATAKGTYGRTRGRATGTRG-3′)

[0041] Synthesized by Shanghai Sangon Company (Sangon Biotech)

[0042] 3. Bacterial genomic DNA extraction:

[0043] Bacterial genomic DNA was extracted using a bacterial DNA extraction kit (TIANamp Bacteria DNA Kit) from Tiangen Biotech and operated ...

Embodiment 2

[0071] The resolution comparison of embodiment 2 CRISPR typing method, MLST and PFGE:

[0072] At the same time, we used two molecular typing methods to type the above 82 strains. Among them, multilocus sequence typing (MLST) is a widely used molecular typing method, and pulsed field gel electrophoresis (PFGE) is currently the gold standard for bacterial molecular typing.

[0073] 1. Salmonella MLST typing:

[0074] The seven housekeeping genes of Salmonella were mainly selected for amplification and sequencing, and the sequence information was uploaded to a special website ( http: / / mlst.ucc.ie / mlst / dbs / Senterica ) to obtain the corresponding MLST type, see the MLST website for specific test operation methods ( http: / / mlst.ucc.ie / mlst / dbs / Senterica / documents / primersEnterica_html )

[0075] PCR conditions:

[0076] Table 3. MLST typing results:

[0077]

[0078]

[0079] 2. Pulsed Field Gel Electrophoresis (PFGE)

[0080] Digest with restriction endonuclease Xba...

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Abstract

The invention provides a salmonella CRISPR (clustered regularlay interspaced short palindromic repeats) sequencing typing method; according to the method, by DNA sequencing of a latest interval sequence between Salmonella CRISPR1 site and CRISPR2 site, CRISPR type of a strain to be detected can be determined, and the Serotype can be predicted. The method is simple, rapid, low-cost and high-resolution, is high in consistency in salmonella serotyping results, and low in requirements on laboratory equipment and software, a new CRISPR type comprises bacterial evolution and regional and other various information, and by combination of a fluorescence probe and other methods, real time and rapid detection can be realized, and the method has popularization and application value.

Description

technical field [0001] The invention relates to a method for molecular typing of Salmonella, belonging to the field of molecular epidemiology. Background technique [0002] Salmonella is an important pathogen that endangers the health of humans and animals. Its genera and types are diverse and its antigens are complex, so the control of Salmonella has caused great difficulties. The molecular typing of Salmonella can be accurately diagnosed and traced. In terms of molecular epidemiology, this convenient, efficient and high-resolution typing method is of great significance for large-scale epidemic detection and the evolution of Salmonella. ;In terms of clinical treatment, since Salmonella has type-specificity in drug resistance and virulence, its accurate typing can largely predict the occurrence, development and outcome of the disease in advance, And it can also provide a reliable basis for clinical treatment and medication. [0003] In addition, outbreak monitoring of Salm...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/42
CPCC12Q1/6858C12Q2531/113C12Q2525/151
Inventor 宋宏彬邱少富李浩李鹏易胜杰谢靖吴志豪郝荣章王立贵柳楠杨超杰
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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