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RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting salmonella paratyphi A

A detection kit, detection reagent technology, applied in DNA/RNA fragments, recombinant DNA technology, microorganism-based methods, etc., to achieve the effects of simple operation, high reliability, and easy observation

Active Publication Date: 2012-08-22
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no application of this method in the detection of Salmonella paratyphi A.

Method used

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  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting salmonella paratyphi A
  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting salmonella paratyphi A
  • RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting salmonella paratyphi A

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Design of primers

[0039] 1. Specific DNA sequence search

[0040] The genome sequences of multiple strains of Salmonella paratyphi A were retrieved from GenBank, and the sequence homology analysis was carried out by BLAST software to find the specific conserved target sequence of the hsdM gene for RT-LAMP primer design. The specific conserved target sequence is shown as SEQ ID Nucleotide sequence shown in No.7.

[0041] 2. Design of primers

[0042] Design six primers for the target sequence, including two inner primers (FIP and BIP), two outer primers (F3 and B3) and two loop primers (LF and LB), the nucleotide sequences of which are as follows:

[0043] F3: 5'-CCTGTTAGAAGTGTTTACAACTT-3';

[0044] B3: 5'-GCTAAACCACCAAATTGTGT-3';

[0045] FIP: 5'-CCAATAGAAATCCTCGGCCAG-

[0046] CCGAGTTTTGATAAGGATGATTG-3';

[0047] BIP: 5'-AACCCTTCAAAAACTACCAAGTCC-

[0048] GAATCTTTAACACGCAACTTTC-3';

[0049] LF: 5'-TGAGGTTGGATTCCAT-3'

[0050] LB: 5'-ATTGA...

Embodiment 2

[0051] Example 2 Establishment of RT-LAMP detection method for Salmonella paratyphi A

[0052] 1. Preparation of simulated blood samples and RNA extraction

[0053] Take freshly cultured bacteria for 4-6 hours (final colony count is 8.0×10 8 cfu / ml), a 10-fold gradient serial dilution of 10 6 ~10 1 cfu / ml, take 3.3 ml of bacterial solution of each dilution and mix it with the same volume of fresh human anticoagulated blood, place it at room temperature for 30 minutes, and use it as a simulated whole blood sample. UCP PurePathogen Blood fieldtest Kit (QIAGEN) was used to extract Salmonella paratyphi A RNA from the above simulated samples, and the specific operation steps were strictly followed the instructions. At the same time, RNA extraction was performed in parallel with blood without bacteria added as a negative control. The final extracted RNA was dissolved in 50 μl of RNase-free sterile pure water, and 5 μl of it was used as a template for RT-LAMP. At the same time, ...

Embodiment 3

[0068] Example 3 RT-LAMP detection kit characteristic evaluation

[0069] 1. RT-LAMP amplification specificity and sensitivity

[0070] Utilize the kit of the present invention to carry out the RT-LAMP detection (see Table 1) of 48 kinds of 136 strains of bacteria altogether, wherein 48 strains of Salmonella paratyphi A are all positive. The other 34 serotypes of Salmonella were amplified negative. Other common diarrhea pathogens (Vibrio cholerae, Vibrio parahaemolyticus, Shigella, diarrhea-causing Escherichia coli) were also amplified negative. Moreover, other common clinical fever pathogens including Staphylococcus aureus, Streptococcus pneumoniae, Borrelia burgdorferi, Leptospira, Legionella pneumophila, Neisseria meningitidis, Rickettsia, and Brucella all amplified Negative, indicating that the RT-LAMP primers screened in this study and the detection kit assembled from these primers have high strain and serotype specificity and sensitivity, both reaching 100%.

[0071] ...

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Abstract

The invention relates to the field of biotechnology, in particular to an RT-LAMP (Reverse Transcription Loop-Mediated Isothermal Amplification) kit for detecting salmonella paratyphi A (S. Paratyphi A). The kit comprises six RT-LAMP primers, wherein the six RT-LAMP primers form a detection system with RT-LAMP reaction solution together; and the nucleotide sequences of the six RT-LAMP primers are shown by SEQ ID No. 1-6. According to the kit, the present epidemic salmonella paratyphi A can be quickly and sensitively detected; and in total RNA (Ribonucleic Acid) detection of pure bacteria, the lowest detection limit is 50 pg per reaction. The kit has the advantages of simplicity in operation, easiness for observation of a reaction result, high specificity, high suitability for detection of export quarantine, food hygiene and clinical specimens and easiness for large-range promotion and application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a reverse transcription-loop-mediated isothermal amplification technique (RT-LAMP) kit for detecting Salmonella paratyphi A. Background technique [0002] Salmonella paratyphi A (S.Paratyphi A) is the pathogenic bacterium that causes paratyphoid fever. The clinical symptoms of paratyphi A and typhoid fever are very similar, and it is difficult to distinguish them. Both of them have sudden onset of high fever as the main symptom. Systemic infectious disease. The disease is highly contagious, has a long course of disease, and can recur repeatedly. Patients need to be hospitalized for isolation and treatment. It is one of the Class B infectious diseases reported in my country's "Law on the Prevention and Control of Infectious Diseases", and it is also a common public health problem faced by the world, especially developing countries. question. [0003] At present, the traditional detec...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/42
Inventor 闫梅英樊粉霞阚飙
Owner ICDC CHINA CDC
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