LAMP detection primer for Nosema ceranae and detection kit employing LAMP detection primer

A technique for detection of microsporidia and primers, which is applied in the biological field, can solve the problems of many operating steps of the oriental honeybee microsporidia, unfavorable widespread promotion and use, and unsuitable detection by grassroots units, so that the reaction results are easy to judge and easy to promote on a large scale Application and meeting the effect of disease surveillance

Inactive Publication Date: 2016-07-13
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

However, the PCR primers designed using this gene have many steps for the detection of Microsporidia orientalis, and it takes a long ti

Method used

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  • LAMP detection primer for Nosema ceranae and detection kit employing LAMP detection primer
  • LAMP detection primer for Nosema ceranae and detection kit employing LAMP detection primer
  • LAMP detection primer for Nosema ceranae and detection kit employing LAMP detection primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The preparation method of the LAMP visual rapid detection kit of Microsporidia orientalis, the specific method steps are as follows:

[0053] S1. Retrieve the RPB1 sequences of N.ceranae and N.apis from the GenBank database, compare the sequences with DNAMAN8 software, and then use the online software: PrimerExploreV4(http: / / primerexplorer.jp / elamp4.0.0 / index.html) designed primers and commissioned the company to synthesize a primer set for LMAP detection; the primer set included the inner primer Nosc-B3: 5'-ATGGAATTCCTGTAAAACGAA-3', and the primer sequence was as shown in SEQ ID NO.1 Nosc-F3: 5'-TATGCACCCTTTCACCAT-3', the primer sequence is shown in SEQ ID NO.2; the outer primer Nosc-BIP: 5'-AAGGAATGGGACTTGTTGCGT-AAAATAACTTTTCCATCACTGTC-3', the primer sequence is shown in SEQ ID NO.3; Nosc-FIP: 5'-ACAGGCTGTTTTATTTCCACATCC-AAGTGTTTGTGAGGGAGAA-3', the primer sequence is as SEQ ID NO.4.

[0054] The target gene sequence and location information of the primers are attach...

Embodiment 2

[0068] Apply the No. orientalis Apis mellifera LAMP visual rapid detection kit prepared with reference to Example 1 for detection, and the specific steps are as follows:

[0069] S1. Prepare nucleic acid samples to be detected:

[0070] Randomly pick 30 colonies of Italian honeybees from the teaching apiary of Fujian Agriculture and Forestry University, and randomly pick 15 worker bees from each box at the nest door, and then prepare according to Example 1.

[0071] S2. Establish a reaction system

[0072] Operate according to the components of the reaction system in Example 2.

[0073] Suggestion: For N samples, (N+3) times the volume of the reaction solution (including a blank control, a negative control, and a positive control should be prepared to avoid errors caused by the cost of aliquoting) should be prepared to ensure that each reaction is evenly aliquoted.

[0074] S3. Adding samples: Mix the reaction solution thoroughly, and then add 2 μL of deionized water (blank co...

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Abstract

The invention discloses an LAMP detection primer for Nosema ceranae and a detection kit employing the LAMP detection primer. The LAMP detection primer comprises outer primers Nosc-F3/Nosc-B3 and inner primers Nosc-FIP/Nosc-BIP, of which the sequences are shown in SEQ ID NO.1-4. The LAMP detection method for Nosema ceranae and the detection kit employing the LAMP detection primer are established by the primer, and the detection kit comprises a primer group, a reaction buffer solution, a positive reference substance, a negative reference substance, a color development solution, BstDNA polymerase and sterile deionized water. The detection kit can quickly and flexibly detect Nosema ceranae, is simple and convenient to operate, low in cost, free from special complicated instruments, easy in reaction result determination and strong in specificity, can better meet the crying needs for illness monitoring, on-site emergency and production sample detection and is easy for wide-range popularization.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to a LAMP detection primer for Microsporidia orientalis and a detection kit thereof. Background technique [0002] Microsporidia orientalis is an important fungal pathogen of honeybees, which widely parasitizes the midgut tissue of honeybees and causes honeybees, especially Western honeybees, to suffer from microsporidiosis. Production of bee products. At the same time, there is still no effective medicine for preventing and treating honeybee microsporidiosis. [0003] In scientific research and beekeeping production, the diagnosis is made through the behavior of honeybees infected with Microsporidia and the changes of midgut tissue morphology and microscopic examination. The results are relatively lagging and unreliable, the detection rate is low, and it is difficult to distinguish between the two spores ( For example, microscopic examination may be confused with yea...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2531/119C12Q2545/113
Inventor 梁勤席伟军李江红陈大福郭睿
Owner FUJIAN AGRI & FORESTRY UNIV
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