Fluorogenic quantitative PCR detection kit for melissococcus pluton and detection method thereof

A detection kit and fluorescence quantitative technology, applied in the direction of microorganism-based methods, microorganism measurement/inspection, biochemical equipment and methods, etc., can solve the problems of detection methods that have not been reported, and achieve simple operation, high sensitivity, good repeatability

Inactive Publication Date: 2017-05-31
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the fluorescent quantitative PCR kit and its detection method for the detection of hive cocci

Method used

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  • Fluorogenic quantitative PCR detection kit for melissococcus pluton and detection method thereof
  • Fluorogenic quantitative PCR detection kit for melissococcus pluton and detection method thereof
  • Fluorogenic quantitative PCR detection kit for melissococcus pluton and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] A fluorescent quantitative PCR detection kit for Melissa officinalis, comprising a forward primer F, a reverse primer R and a TaqMan probe P, wherein the sequence of the forward primer F is 5'-AACGCTTCTTCTGATTAAG-3', and the sequence of the reverse primer R is 5' '-GCCTTTTAACTCCCTCTTC-3'; TaqMan probe P sequence is 5'-FAM-ACGGTATTAGCACCTGTTTCCAAAT-BHQ1-3'. The kit includes the following reagents (50 reactions):

[0033] (1) Positive standard: Use F and R primers to amplify the genomic DNA of Melissa melissae, clone the product into the pMD-18T vector, transform DH5α Escherichia coli, extract the positive clone plasmid by alkaline lysis, and calculate the purity and concentration of the plasmid. 10-fold serial dilution to 1000 copies / μL as a positive standard, 100 μL / vial, stored at -20 °C;

[0034] (2) Negative control: 1 negative control with a concentration of 100 ng / μL in 100 μL, and the total DNA extracted from healthy bee tissues not infected with Melilococcus hiv...

Embodiment 2

[0039] The detection method using the honeycomb melissa officinale fluorescent quantitative PCR detection kit comprises the following steps:

[0040] (1) PCR reaction system configuration: Add 17.0 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 5.5 μL of sterilized water to make the total reaction volume 25.0 μL ;

[0041] (2) PCR reaction program: pre-denaturation at 95°C for 3 min; then denaturation at 95°C for 5 s, annealing and extension at 59°C for 30 s, a total of 40 cycles, and single-point fluorescence detection at 59°C;

[0042] (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 s...

Embodiment 3

[0044] Establishment of Standard Curve for Fluorescent Quantitative PCR Detection Kit of Apis coccus in Apiary

[0045] (1) Standard DNA dilution: Dilute the positive clone plasmid containing the target fragment 10 times with sterile water to 5×10 8 Copies / μL ~5×10 1 A range of DNA standards in copies / µL.

[0046] (2) Establishment of the standard curve equation: the diluted DNA standard was used as the template, and each standard sample was treated 3 times, and the total DNA of healthy bee tissues not infected with Melilococcus hives was used as the control, and sterile water was used as the blank control . The fluorescent quantitative PCR reaction system of 25 μ L is configured in the manner described in Example 2 and carried out fluorescent quantitative PCR reaction; figure 2 The amplification curve shown is then plotted as image 3 For the standard curve shown, construct the standard curve equation. The standard curve equation constructed in this embodiment is Y=-3.3...

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Abstract

The invention relates to a fluorogenic quantitative PCR detection kit for melissococcus pluton and a detection method thereof. The kit is especially used for detecting melissococcus pluton. The kit comprises (1) a positive standard substance, (2) a negative control, (3) a PCR reaction solution, (4) ExTaq enzyme and (5) aquae sterilisata. The invention has the advantages of (1) high stability and specificity: high specificity in detection for melissococcus pluton, no cross reaction with other bee pathogenic bacteria and excellent repeatability, (2) high sensitivity capable of reaching up to 50 copy/uL and (3) simple, convenient and quick operation: capability of completing the whole reaction within 2 hours. The kit provided by the invention can be used for diagnosing bee European foul brood and melissococcus pluton and has significance in early prevention and timely treatment of the diseases.

Description

technical field [0001] The invention relates to a fluorescent quantitative PCR detection kit for honeybee melissa officinalis and a detection method thereof, which belongs to the technical field of animal health and is suitable for the diagnosis and monitoring of European larval rot disease of bees and the detection of honeybee honeybee coccus in bee products. Background technique [0002] European foulbrood (EFB for short) is a bacterial, acute, and devastating infectious disease caused by Melissa coccus in the hive. It can lead to the death of a large number of bee larvae, and eventually lead to the demise of the entire bee colony, causing great harm. The disease is an infectious disease of the digestive tract, which mainly affects bee larvae of 1 to 2 days old. After an incubation period of 2 to 3 days, most of them die when they are not sealed at the age of 3 to 4 days. [0003] EFB occurs all over the world, among which the incidence of Chinese bees is more severe than ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 张体银郑腾李丹丹白泉阳王武军张志灯于师宇
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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