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Simultaneous amplification and testing reagent kit for VC (vibrio cholerae)

A Vibrio cholerae, real-time fluorescence technology, applied in the field of probes and related kits, primers, can solve the problems of false negative detection cost, low sensitivity, false positive test results, etc.

Active Publication Date: 2012-10-10
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In order to solve the problems that the existing Vibrio cholerae (VC) detection method has low sensitivity, long detection cycle, easy to cause contamination of amplified products, false positive or false negative of experimental results and high detection cost, the present invention provides a A real-time fluorescent nucleic acid constant temperature amplification detection technology for Vibrio cholerae (VC) with short detection cycle, high sensitivity, high specificity, low pollution, stable reaction and low detection cost, easy to popularize and apply, including special primers, probes, reagents box and its use

Method used

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  • Simultaneous amplification and testing reagent kit for VC (vibrio cholerae)
  • Simultaneous amplification and testing reagent kit for VC (vibrio cholerae)
  • Simultaneous amplification and testing reagent kit for VC (vibrio cholerae)

Examples

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Effect test

Embodiment 1

[0098] Embodiment 1, the design of special primers and probes for detection of Vibrio cholerae (VC) by real-time fluorescent nucleic acid constant temperature amplification

[0099] The present invention selects no secondary structure and highly conserved segment in VC bacteria toxR as the amplified target sequence region (its nucleotide sequence is shown in sequence 1 in the sequence table), and according to the principle of primer probe design, DNA ATAR, DNAman Software and artificially designed special primers and probe sequences for real-time fluorescent nucleic acid constant temperature amplification detection of Vibrio cholerae (VC), and obtained the following specific sequences:

[0100] (1) A capture probe (TCO, Target Capture Oligo) that can specifically bind to the target nucleic acid (VC RNA) sequence of Vibrio cholerae (VC) as shown in Sequence 1 in the sequence listing, the core of the capture probe The nucleotide sequence is 5'-GCCAGCUGGUAUCUUCGACUGACUUCAAAAAAAAA...

Embodiment 2

[0104] Embodiment 2, prepare the real-time fluorescent nucleic acid constant temperature amplification detection kit of Vibrio cholerae (VC)

[0105] Using the special primers and probes provided in Example 1, the real-time fluorescent nucleic acid constant temperature amplification detection kit for Vibrio cholerae (VC) of the present invention was obtained. The kit includes capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, VC detection probe, internal standard detection probe, M-MLV reverse transcriptase and T7 RNA polymerase.

[0106] The capture probe exists in the nucleic acid extraction solution, the T7 primer, nT7 primer, VC detection probe, and internal standard detection probe exist in the VC detection solution, and the M-MLV reverse transcriptase and T7 RNA polymerase It exists in the SAT enzyme solution. Specifically, the kit is divided into A box (specimen processing unit) stored at 2-30°C and B box (nucleic acid amplification detection unit) stored...

Embodiment 3

[0139] Embodiment 3, detection sensitivity of real-time fluorescent nucleic acid constant temperature amplification of Vibrio cholerae

[0140] Detect Vibrio cholerae (VC) in the food sample with the kit of the present invention (see Example 2 for the composition, there is no VC internal standard and the internal standard detection probe in the detection solution in the kit), and the specific method comprises the following steps:

[0141] (1) Bacterial solution dilution

[0142] The measured concentration is 1×10 5 CFU / mL Vibrio cholerae culture, 10-fold serial dilution to 10cFU / mL as Vibrio cholerae linear sensitivity reference.

[0143] (2) Nucleic acid extraction

[0144] 2.1 Add 200 μl lysate (containing HEPES 35mM, (NH 4 ) 2 SO 4 20 mM), 200 μl of bacterial liquid, and use the lysate to lyse the Vibrio cholerae (VC) in the sample to be tested to obtain a lysate containing the Vibrio cholerae (VC) nucleic acid.

[0145] 2.2 Add 100μl nucleic acid extraction solution ...

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Abstract

The invention discloses a simultaneous amplification and testing reagent kit for VC (vibrio cholerae). The simultaneous amplification and testing reagent kit for the VC comprises a capture probe, a VC testing primer T7, a VC testing primer nT7, a VC testing probe, an M-MLV reverse transcriptase, a T7 RNA (ribonucleic acid) polymerase and the like. The simultaneous amplification and testing reagent kit for the VC can test VC RNA in food, has the advantages of high specificity, high sensitivity (capable of reaching 100 CFU / ml), low contamination (an amplified product RNA is easy to degrade in the natural environment) and rapidness in testing (testing can be completed within 50 minutes conventionally), plays an important role in rapid testing of the VC and is wide in application prospect.

Description

technical field [0001] The invention relates to the biological detection technology of pathogenic bacteria, in particular to the primers and probes used in the real-time fluorescent nucleic acid constant temperature amplification detection of Vibrio cholerae (VC) which combines the specific target capture technology and the real-time fluorescent nucleic acid constant temperature amplification detection technology. Needles and related kits. Background technique [0002] Vibrio cholerae (V.cholera) is the pathogen of human cholera, which is one of the ancient and widespread severe infectious diseases. It has caused many pandemics in the world, mainly manifested as severe vomiting, diarrhea, dehydration, and high mortality rate. It belongs to international quarantine infectious diseases. Human beings are the only susceptible persons to Vibrio cholerae under natural circumstances, mainly through oral infection through contaminated water sources or food, especially aquatic produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/63
CPCY02A50/30
Inventor 李豪张长明于明辉居金良
Owner SHANGHAI RENDU BIOTECH
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