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Fluorescent quantitative PCR (polymerase chain reaction) primer for rapidly diagnosing nosema antheraeae and application of primer

A fluorescence quantitative and microsporidia technology, which is applied in the direction of microorganism-based methods, microorganism measurement/inspection, microorganisms, etc., can solve the problems of low detection sensitivity, cumbersome operation, and late detection period of the tussah silkworm microsporidia diagnostic technology. Achieve the effect of eliminating false positives in molecular diagnosis, quantifying reliability, and improving yield

Pending Publication Date: 2016-12-14
辽宁省农业科学院大连生物技术研究所
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Problems solved by technology

[0005] The purpose of the present invention is to provide a group of fluorescence quantitative PCR primers and its application for rapid diagnosis of tussah microsporidia for the problems of low detection sensitivity, cumbersome operation and late detection period of the existing tussah microsporidia diagnostic technology

Method used

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  • Fluorescent quantitative PCR (polymerase chain reaction) primer for rapidly diagnosing nosema antheraeae and application of primer
  • Fluorescent quantitative PCR (polymerase chain reaction) primer for rapidly diagnosing nosema antheraeae and application of primer
  • Fluorescent quantitative PCR (polymerase chain reaction) primer for rapidly diagnosing nosema antheraeae and application of primer

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Experimental program
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Embodiment 1

[0041] The specificity of embodiment 1 conventional PCR identification primer

[0042] Take the tussah microsporidia suspension, centrifuge at 5000r / min for 5min, remove the supernatant and retain the spore precipitate, add the extract (10mmol / L Tris-HCL pH 8.0; 10mmol / L EDTA pH 8.0; 100mmol / L NaCl; 2% SDS; 0.039 mol / L DTT), plus proteinase K (final concentration: 100 μg / mL), digested at 55°C for 2 hours, extracted with phenol / chloroform, and obtained the genomic DNA of Nos. tussah mori as a positive reference.

[0043] Take another tussah silkworm chrysalis, tussah silkworm larvae and tussah silkworm mother moth infected with microparticles disease, take appropriate amount of tussah silkworm pupae tissue, tussah silkworm larvae tissue, tussah silkworm moth tissue and tussah silkworm eggs (taken from the body of tussah silkworm moth), and put them into No. 1# respectively. In the 1.5mL EP tubes of 2#, 3# and 4#, take an appropriate amount of healthy tussah silkworm chrysalis t...

Embodiment 2

[0049] Standard curve and primer amplification efficiency of embodiment 2 fluorescence quantitative PCR

[0050] Preparation of standards:

[0051] The positive reference PCR product in Example 1 was recovered and purified, connected to the pMD-18T vector, transformed into DH5α competent cells, spread on a plate medium for cultivation, and picked positive colonies to extract plasmid DNA for sequencing identification. Quantify the pMD-18T / Np-SSUrRNA plasmid DNA with correct sequencing results, convert it into copy number, and dilute it in a 10-fold gradient with sterile water to 1×10 8 ~1×10 1 copies / μL of the standard.

[0052] Drawing of standard curve and determination of detection limit and primer efficiency:

[0053] ABI 7300 fluorescent quantitative PCR instrument was used for amplification. Fluorescence quantitative PCR reaction system: SYBR PremixExTaq 10 μ L, ROX Reference Dye I 0.4 μ L, the upstream primer (10pmol / μL) 0.2 μ L as described in SEQ ID NO:1, the downs...

Embodiment 3

[0055] Embodiment 3 Fluorescent quantitative PCR detects Microsporidia tussah

[0056] Take tussah silkworm chrysalis, tussah silkworm larvae and tussah silkworm mother moth infected with microparticles disease, take appropriate amount of tussah silkworm pupae tissue, tussah silkworm larvae tissue, tussah silkworm moth tissue and tussah silkworm eggs (taken from the body of tussah silkworm moth), and put them into No. 1 and 2 respectively. In #, 3# and 4# 1.5mLEP tubes, take an appropriate amount of healthy tussah silkworm chrysalis tissue into the negative reference EP tube, add nucleic acid extraction solution (10mmoL / L Tris-HClpH8.0, 100mmoL / L EDTA, 0.5% SDS, 100μg / mL proteinase K), digested at 55°C for 4 hours, extracted with phenol / chloroform, and obtained the DNA of each sample.

[0057] For 1-4# sample DNA, deionized water, healthy pupae DNA and positive reference as template DNA, ABI 7300 fluorescent quantitative PCR instrument was used for fluorescent quantitative PC...

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Abstract

The invention discloses a specific primer for diagnosing nosema antheraeae and an application of the primer. High-sensitivity specific detection for the nosema antheraeae is realized by an SYBR Green fluorescent quantitative PCR (polymerase chain reaction) method, the optimal reaction condition includes 95 DEG C and 30 seconds, circulation is performed for 40 times under the condition of 95 DEG C and 5 seconds and 62.5 DEG C and 30 seconds, and the melting curve condition includes 95 DEG C, 15 seconds, 60 DEG C, 60 seconds, 95 DEG C, 15 seconds, 60 DEG C and 15 seconds. Detection sensitivity is improved by two magnitude orders as compared with that of disclosed PCR technology, and lower detection limit includes that each reaction system contains 0.0046ng nosema genome DNA (deoxyribonucleic acid). Reliability of detection results is quantized by calculating primer amplification efficiency. The specific primer effectively solves the problems of low sensitivity, late detection period, easiness in leak detection, environmental pollution caused by conventional PCR nucleic acid coloring agents and the like, and has a wide application prospect in terms of antheraeae pebrine inspection and quarantine and cocoon quality improvement.

Description

technical field [0001] The invention belongs to the field of molecular biology detection of tussah microsporidia, in particular to a pair of specific primers and its application in the detection of tussah microsporidia. Background technique [0002] Tussah microsporidiosis, also known as tussah microsporidiosis, commonly known as rust, slag disease, root disease, black slag disease, dry tiger, etc., is a chronic infectious disease caused by tussah microsporidia parasitism. In the first year of Xuantong (1909), Xu Lan's "Simplified Method of Tussah Silkworm" also mentioned "black spot disease of tussah silkworm". Tussah microparticle disease occurs in all stages of tussah silkworm development, and has a wide distribution. It occurs in tussah silkworm areas in Heilongjiang, Jilin, Liaoning, Shandong, Henan, Guizhou and other provinces in China. It is one of the main diseases of tussah silkworm production. The incidence rate of silk cocoon production in Liaoning Province is 30...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6893C12Q1/6851C12Q2531/113C12Q2563/107
Inventor 米锐李佩佩范琦李亚洁赵振军李树英都兴范
Owner 辽宁省农业科学院大连生物技术研究所
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