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 micr

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

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0041] Example 1 Conventional PCR to identify the specificity of primers

[0042] Take the Nosema tumefaciens 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; 0.039mol / L DTT), plus proteinase K (final concentration 100μg / mL), digested at 55°C for 2 hours, and extracted with phenol / chloroform to obtain the genomic DNA of Nosema tussah as a positive reference.

[0043] In addition, take the tussah pupa, tussah larvae and tussah female moth infected with particulate disease, and take the tussah pupa tissue, tussah larva tissue, tussah moth tissue and tussah silkworm eggs (taken into the body of the tussah silkworm). 2#, 3# and 4# 1.5mL EP tube, take healthy tussah silkworm pupa tissue into the negative reference EP tube, add nucleic acid extraction solution (10mmoL / LTris-HCl pH8.0, 100mmoL / L EDTA, 0.5% SDS , 100μg / mL proteinase K), digested a...

Example Embodiment

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

[0050] Preparation of standards:

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

[0052] The drawing of standard curve and the determination of lower detection limit and primer efficiency:

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

Example Embodiment

[0055] Example 3 Fluorescence quantitative PCR detection of Nosema tussah

[0056] Take tussah silkworm pupa, tussah larvae and tussah female moth infected with particulate disease, take tussah pupa tissue, tussah larva tissue, tussah moth tissue and tussah silkworm eggs (taken into the body of the tussah silkworm), and put them into number 1#, 2 #, 3# and 4# 1.5mL EP tube, take healthy tussah silkworm pupa 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, and extracted with phenol / chloroform to obtain each sample DNA.

[0057] Using 1-4# sample DNA, deionized water, healthy pupal DNA and positive reference as template DNA, use ABI 7300 fluorescent quantitative PCR instrument to perform fluorescent quantitative PCR. Configuration reaction system: SYBR Premix ExTaq 10μL, ROX ReferenceDye I 0.4μL, the upstream primer (10pmol / μL) as described in SEQ...

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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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