Real-time fluorescent PCR detection kit for Nosema bumblebee and detection method thereof

A detection kit and technology for microsporidia, which is applied in the field of animal health, can solve the problems that the detection method of bumblebee microsporidia has not been reported, and achieve the effects of high sensitivity, good repeatability, and low fluorescence background

Pending Publication Date: 2019-01-18
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows scientists to accurately identify bumblefele organisms by measuring their DNA sequences or comparing them against known samples from databases that have been previously identified. By selecting certain fragments of these genes instead of others, this method provides more stable results compared to existing methods such as qPCR. Additionally, it simplifies analysis procedures while maintaining accuracy over multiple generations. Overall, this new technique offers technical benefits including improved safety measures due to its ability to monitor both biological processes at once (fluorogenesis) without requiring expensive equipment like laser scanning microscopies).

Problems solved by technology

The problem addressed in these patents relates to identifying harmful organisms called Micromaspora sppimberma, which causes damage to many commercial crops worldwide including Beehive industry. Current technologies involve immunochemistry, histoplasmic shaking culture, flow cytometry, X-ray analysis, and others like qPCFISH. These existing approaches suffer drawbacks due to lack of effective target sequences, poor reproduction efficiency, difficulty in obtaining sufficient samples, difficulties in isolating pure strains during mass spectra imagery processing, and potential issues related to gene mutation caused by environmental factors.

Method used

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  • Real-time fluorescent PCR detection kit for Nosema bumblebee and detection method thereof
  • Real-time fluorescent PCR detection kit for Nosema bumblebee and detection method thereof
  • Real-time fluorescent PCR detection kit for Nosema bumblebee and detection method thereof

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Effect test

Embodiment 1

[0035] A real-time fluorescent PCR detection kit for Microsporidium bumblebee, comprising forward primer N.bombi-F, reverse primer N.bombi-R and TaqMan probe N.bombi-P, wherein forward primer N.bombi-F The sequence of the reverse primer N.bombi-R is 5'-GTCTCTCAGGCTCCTTCTCC-3', the sequence of the reverse primer N.bombi-R is 5'-ATGTGCACCGCAGATAACTA-3'; the sequence of the TaqMan probe N.bombi-P is 5'-FAM-ACCGTTACCCGTCACTGCCTTGT-BHQ1-3'. The kit includes the following reagents (50 reactions):

[0036] (1) Positive standard: Use primers N.bombi-F and N.bombi-R to amplify the genomic DNA of No. bumblebee, clone the product into pMD-18T vector, transform DH5α Escherichia coli, and extract positive clones by alkaline lysis Plasmid, after calculating the plasmid purity and concentration, 10-fold serial dilution to 10,000 copies / μL as a positive standard, 100 μL / cartridge, stored at -20 °C;

[0037] (2) Negative control: 1 negative control with 100 μL concentration of 100 ng / μL, the ...

Embodiment 2

[0042] Optimization of the annealing temperature for the detection method of the real-time fluorescent PCR detection kit for No. bumblebee

[0043] (1) PCR reaction system: Add 16.5 μL of PCR reaction solution, 0.5 μL of 5 U / μL ExTaq enzyme, and 2.0 μL of positive control to each PCR tube, and then add 60. μL of sterilized water to make the total reaction volume 25.0 μL µL;

[0044] (2) PCR optimization reaction program: pre-denaturation at 95°C for 5 min; then denaturation at 95°C for 5 s, annealing at 55°C-65°C (gradient temperature) for 30 s, and 3 repetitions for each gradient temperature, a total of 40 cycles;

[0045] (3) Selection of annealing temperature: the optimization results of annealing temperature are as follows: figure 2 As shown, the fluorescence intensity of the amplified product was higher when the annealing temperature was 59.0°C and 61.4°C than at other temperatures, so 60°C was selected as the annealing temperature for the reaction program of the kit. ...

Embodiment 3

[0047] The detection method using the real-time fluorescent PCR detection kit of Bombus bumblebee comprises the following steps:

[0048] (1) PCR reaction system configuration: Add 16.5 μL of PCR reaction solution, 0.5 μL of 5 U / μL ExTaq enzyme, 2.0 μL of sample DNA to be tested into the PCR tube, and then add 6.0 μL of sterilized water to make the total reaction volume 25.0 μL ;

[0049] (2) PCR reaction program: pre-denaturation at 95°C for 5 min; then denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, a total of 40 cycles, and single-point fluorescence detection at 60°C;

[0050] (3) Judgment of results: The negative control has no Ct value and no amplification curve, while the positive control has a Ct value ≤ 35 and a typical amplification curve, indicating that the experiment is valid, otherwise the experiment is invalid. If the experiment is valid, the sample to be tested has no Ct value and no amplification curve, indicating that the sample does n...

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Abstract

The invention relates to a real-time fluorescent PCR detection kit for bumblebee microsporidia and a detection method thereof, which are specially used for detecting bumblebee microsporidia. The kit comprises: (1) a positive standard; (2) negative control; (3) PCR reaction solution; (4) ExTaq enzyme; (5) sterile water. The invention has the following advantages: (1) good stability and specificity:detection of bumblebee microsporidia has high specificity, has no cross reaction with other microsporidia and bumblebee pathogens, and has good repeatability; (2) high sensitivity: the sensitivity can reach 20 copies/[mu] L; (3) easy and fast operation: the whole reaction can be completed within 2 hours. The kit can be used for detecting Nosema bombycis and differentiating and diagnosing the Nosema bombycis from other Nosema bombycis, and has important significance for early prevention and timely treatment of the disease. The kit can be used for detecting Nosema bombycis and differentiating and diagnosing the Nosema bombycis from other Nosema bombycis.

Description

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Claims

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Application Information

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Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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