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Primer, probe and reagent for rapidly detecting vibrio parahaemolyticus at room and isothermal temperature, and method realized through reagent

A room temperature isothermal, hemolytic Vibrio technology, applied in biochemical equipment and methods, microbial determination/inspection, resistance to vector-borne diseases, etc. Effect

Inactive Publication Date: 2020-09-04
潍坊安普未来生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These techniques are relatively time-consuming, and the sensitivity to detection of Vibrio parahaemolyticus needs to be further optimized
[0007] In summary, there are obviously inconveniences and defects in the actual use of the existing technology, so it is necessary to improve

Method used

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  • Primer, probe and reagent for rapidly detecting vibrio parahaemolyticus at room and isothermal temperature, and method realized through reagent
  • Primer, probe and reagent for rapidly detecting vibrio parahaemolyticus at room and isothermal temperature, and method realized through reagent
  • Primer, probe and reagent for rapidly detecting vibrio parahaemolyticus at room and isothermal temperature, and method realized through reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Screening of primers

[0053] With reference to the VP tlh gene sequence published on GenBank, the inventors of the present application have carried out relatively in-depth research on the genome and function of TLH of VP. All VP isolates contain thermolabile hemolysin, which is Unique to VP, so its coding gene tlh gene field was used as the target gene to detect VP. A large number of experiments have shown that different primers have a certain influence on the effect and sensitivity of isothermal amplification. Therefore, four pairs of different primers were designed for the tlh gene of VP: VP-1, VP-2, VP-3, VP-4, as shown in Table 1 below.

[0054] Table I

[0055]

[0056] image 3 It is the electrophoretic pattern of the effect of the primers of the present invention on the amplification effect. from image 3 It can be seen from the above that different primers have an impact on the amplification effect, and the brightness of the primer VP-4 is th...

Embodiment 2

[0061] Embodiment 2: Fluorescence detection

[0062] This example is used to illustrate the normal temperature and constant temperature fluorescence reaction performed on the fluorescence instrument.

[0063] 1. According to the published tlh gene sequence of Vibrio parahaemolyticus (VP) on NCBI GenBank, based on the design principles of primers and fluorescent probes, a pair of primers (VP-4-F and VP-4-R) and a fluorescent probe (VP-Probe), the sequences are shown in Table 2:

[0064] Table II

[0065]

[0066] Among them, F is a fluorescent group, H is tetrahydrofuran, B is a quenching group, and the 3' end is labeled with the C3 interarm.

[0067] 2. The amplification reaction system is:

[0068] Table three

[0069]

[0070] *Note: The lyophilized powder reagent contains the necessary enzymes and auxiliary components mentioned above.

[0071] 3. Amplification reaction procedure: 39 degrees constant temperature, 20 minutes.

[0072] The VP template was diluted 10 ...

Embodiment 3

[0073] Example 3: Specificity Test

[0074] It is used to test the specificity of the selected primers, select several other strains, and use the primers VP-4-F, VP-4-R and VP-Probe to detect whether there will be non-specific amplification and interpret false positive results. Vibrio cholerae, Shigella baumannii, Salmonella typhimurium and Pseudomonas aeruginosa were selected respectively, and the VP standard strain ATCC33847 was used as a comparison. The result is as Image 6 As shown in the figure, 1 is the VP standard strain ATCC33847, 2 is Vibrio cholerae, 3 is Shigella baumannii, 4 is Salmonella typhimurium, 5 is Pseudomonas aeruginosa, and NTC is a negative control primer. It can be seen that VP-4-F, VP-4-R and VP-Probe have better specificity and no false positive interpretation results.

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Abstract

The invention is suitable for the technical field of vibrio parahaemolyticus detection, and provides a primer and probe for rapidly detecting vibrio parahaemolyticus at room and isothermal temperature. A reagent and method which include the primer and probe and can rapidly detect the vibrio parahaemolyticus are also provided. Thus, rapid, real-time and specific detection can be performed on the vibrio parahaemolyticus under room and isothermal temperature conditions. Through the combined action of the specific primer, the specific fluorescent probe, four engineering enzymes and other chemicalcomponents, the rapid detection of nucleic acid targets can be realized in an instrument having fluorescence detection functions; closed tube detection can be guaranteed through strict experimental operation steps, so that aerosol pollution can be effectively prevented; and the detection method is suitable for the detection of various fluorescence detection equipment.

Description

technical field [0001] The invention relates to the technical field of Vibrio parahaemolyticus detection, in particular to a primer, a probe, a reagent and a method for rapid isothermal detection of Vibrio parahaemolyticus at room temperature. Background technique [0002] Vibrio Parahemolyticus (VP) belongs to the Vibrio family, Vibrio genus, Gram-negative halophilic bacteria, Polymorphobacteria or Campylobacter spp. It was first isolated by Japanese scholars such as Fujino in 1950 and named as para Pasteurella hemolytica. In 1955, Takikawa et al identified it as a halophilic bacteria due to its salt tolerance, until 1963 Sakazaki et al named it Vibrio parahaemolyticus. [0003] Vibrio parahaemolyticus is a polymorphic bacteria, common arc-shaped, oval, rod-shaped or filamentous, mostly irregularly distributed, occasionally in pairs, with flagella, without spores and capsules. The bacteria are about 1.4-2.4 μm long and 0.5-0.8 μm wide. Vibrio parahaemolyticus has two for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6851C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6851C12Q1/689C12Q2531/119C12Q2521/319C12Q2521/507C12Q2522/101C12Q2537/1376C12Q2521/101C12Q2563/107Y02A50/30
Inventor 曹伟刘琼瑶娄志英宋文凤刘国宪
Owner 潍坊安普未来生物科技有限公司
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