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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  • Abstract
  • Description
  • Claims
  • Application Information

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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

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Example Embodiment

[0038] Example 1 Design of primers

[0039] 1. Search for specific DNA sequence

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

[0041] 2. Design of primers

[0042] Six primers are designed 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 sequence is as follows:

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

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

[0045] FIP: 5’-CCAATAGAAATCCTCGGCCAG-

[0046] CCGAGTTTTGATAAGGATGATTG-3’;

[0047] BIP: 5’-AACCCTTCAAAACTACCAAGTCC-

[0048] GAATCTTTAACACGCAACTTTC-3’;

[0049] LF: 5’-TGAGGTTGGATTCCAT-3’

[0050] LB: 5’-ATTGAGTGGGTTAACAAA-3’

Example Embodiment

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

[0052] 1. Preparation of blood mock specimens 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 serial dilution of 10 6 ~10 1 cfu / ml, take 3.3 ml of each dilution of the bacterial solution and mix it with the same volume of fresh human anticoagulant blood, and place it at room temperature for 30 minutes, as a simulated whole blood specimen. The QIAamp UCP PurePathogen Blood fieldtest Kit (QIAGEN) was used to extract RNA from Salmonella paratyphi A on the simulated specimens, and the specific operation steps were strictly in accordance with the instructions. At the same time, RNA extraction was performed in parallel with blood without bacteria as a negative control. The final extracted RNA was dissolved in 50μl of RNase-free sterile pure water, and 5μl of it was used as a template for RT-LAMP. At the same time, take a ser...

Example Embodiment

[0068] Example 3 Evaluation of RT-LAMP detection kit characteristics

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

[0070] Using the kit of the present invention, a total of 48 kinds of 136 strains of bacteria were detected by RT-LAMP (see Table 1), of which 48 strains of Salmonella paratyphi A were positive. The other 34 serotypes of Salmonella were negative for amplification. Other common diarrhea pathogens (Vibrio cholerae, Vibrio parahaemolyticus, Shigella, diarrheal Escherichia coli) also amplified negative. In addition, other common clinical fever pathogens including Staphylococcus aureus, Streptococcus pneumoniae, Borrelia burgdorferi, Leptospira, Legionella pneumophila, Neisseria meningitidis, Rickettsia, and Brucella were all amplified Negative, indicating that the RT-LAMP primers selected in this study and the detection kit assembled from these primers have high strain and serotype specificity and sensitivity, both reaching 100%.

[0071] Table 1 RT-LAMP...

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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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