Primer group and probe capable of simultaneous quantitative determination of pratylenchus neglectus and pratylenchus thornei Sher&Allen and application thereof

A technique for rejecting Brachypodium nematodes and Sunny Brachyomorpha, which is applied in the direction of determination/inspection of microorganisms, recombinant DNA technology, biochemical equipment and methods, etc.

Active Publication Date: 2015-08-12
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to overcome the deficiencies of the existing detection techniques of the brevity nematodes and the brevity sonny, especially the deficiency of the simultaneous detection technology of the two kinds of nematodes. and Brachybody sonny elegans provide a set of primer sets and probes

Method used

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  • Primer group and probe capable of simultaneous quantitative determination of pratylenchus neglectus and pratylenchus thornei Sher&Allen and application thereof
  • Primer group and probe capable of simultaneous quantitative determination of pratylenchus neglectus and pratylenchus thornei Sher&Allen and application thereof
  • Primer group and probe capable of simultaneous quantitative determination of pratylenchus neglectus and pratylenchus thornei Sher&Allen and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Primer and probe design

[0041] 1、根据NCBI中多种短体线虫的28s核糖体DNA序列(基因登录号为:EU130854、JX046968、KM200579、JQ303333、JX261951、JX261960、JX261954、EU130875、EU130866、EU130873、EU130845、JN244270、EU130889、HQ662581、 JN091970, AM231916, JX261948, KF765435, DQ498832, JX047005, JQ003994, GU214114, JX144360 ), design primers and probes as follows:

[0042] (1) Unselected short-body nematodes:

[0043] Upstream primer pnF (as shown in SEQ ID NO.1):

[0044] 5'-GAACCCGAGGTGGGCTAGC-3'.

[0045] Downstream primer pnR (as shown in SEQ ID NO.2):

[0046] 5'-GGCAGGACCGAACCTGCGA-3'.

[0047] Specific probe prbpn (as shown in SEQ ID NO.3):

[0048] 5'-FAM-TTCATTCCCTGGGGCATGGCTTACG-ECL-3'.

[0049] (2) Sonny brevis:

[0050] Upstream primer ptF (as shown in SEQ ID NO.4):

[0051] 5'-AGTGCCATTCGCCTTGGGC-3';

[0052] Downstream primer ptR (as shown in SEQ ID NO.5):

[0053] 5'-CTCTTACACTGAGCCTGGGA-3';

[0054] Specific probe prbpt (as shown in SEQ ID NO.6):

[0055] 5'-ROX-ATGGCTTAC...

Embodiment 2

[0068] Example 2 Optimizing the reaction system of multiplex real-time fluorescent PCR

[0069] In the multiplex PCR system, there will be mutual interference between different primer combinations, therefore, the concentration and ratio of primers and probes need to be optimized.

[0070] 1. Primer concentration optimization

[0071] (1) qPCR reaction conditions

[0072] Different ratios of Pn and Pt specific primers were used to carry out PCR amplification using the DNA of the unselected Brachybody elegans and Brachybody sonny elegans as templates, respectively, and the optimal ratio of Pn and Pt specific primers was screened.

[0073] The PCR reaction system is: 1 μL DNA, 0.16-0.4 μM for the upstream and downstream primers PnF / PnR, 0.16-0.4 μM for the upstream and downstream primers PtF / PtR, 10 μL SYBR ? Green Realtime PCR Master Mix and excess ddH 2 O, a total of 20 μL.

[0074] where the SYBR ? Green Realtime PCR Master Mix was purchased from Toyobo (Shanghai) Biote...

Embodiment 3

[0090] Embodiment 3 multiplex real-time fluorescent PCR method

[0091] Based on the above-mentioned research, the multiple real-time fluorescent PCR method that qualitative and quantitative detection of the simultaneous qualitative and quantitative detection of the present invention is as follows:

[0092] PCR reaction system: 1 μL DNA, 0.16 μM pn primer, 0.2 μM pt primer concentration, 0.10 μM probe prbpn concentration, 0.08 μM probe prbpt concentration, 10 μL real-time fluorescent quantitative PCR premix reagent and remaining ddH 2 O, a total of 20 μL.

[0093] Wherein, the real-time fluorescent quantitative PCR premix reagent is Realtime PCR Master Mix, purchased from Toyobo (Shanghai Biotechnology Co., Ltd.), which already contains DNA polymerase, buffer and dNTP required for real-time fluorescent quantitative PCR reaction.

[0094] PCR reaction program: pre-denaturation at 95°C for 2min; 95°C for 15s, annealing at 65°C for 15s and 72°C for 30s, a total of 40 cycles. ...

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Abstract

The invention discloses a primer group and probe capable of simultaneous quantitative determination of pratylenchus neglectus and pratylenchus thornei Sher&Allen, the primer group includes a primer pair pnF / pnR and a primer pair ptF / ptR, and the probe is prbpt and prbpn. The sequences of the primer pnF / pnR are respectively as shown in SEQ ID NO.1 and 2, and the sequences of the primer ptF / ptR are respectively as shown in SEQ ID NO.4 and 5, and the sequences of the prbpt and prbpn are respectively as shown in SEQ ID NO. 3 and 6. Multiplex real-time fluorescence PCR method established by the primer group and the probe can not only specifically simultaneously determine the pratylenchus neglectus and pratylenchus thornei Sher&Allen from a mixed nematode sample or matrix, effectively overcomes the disadvantages of heavy workload and high detection cost, and can achieve the detection sensitivity of a single nematode. At the same time, the method can also be used for quantitative analysis of the pratylenchus neglectus and pratylenchus thornei Sher&Allen, and has important propulsion significance to actual testing work.

Description

technical field [0001] The invention belongs to the technical field of plant pathogen detection. More specifically, it relates to a primer set and a probe capable of simultaneously quantitatively detecting Brachybody nematode unselected and Brachybody sonnya and applications thereof. Background technique [0002] Brachybody nematodes, also known as root-rot nematodes, are an important migratory endoparasitic nematode consisting of 68 valid species. Root rot nematodes move, puncture and feed in the root of the plant, which will cause the formation of necrotic spots and cavities in the root tissue, which will cause root tissue necrosis. Plants infested by root rot nematodes show symptoms of water and nutrient deficiencies due to root necrosis. Brachybody nematodes are one of the three plant nematodes that cause the greatest economic losses, among which Brachybody nematodes ( Pratylenchus neglectus ) and Brachybody sonny ( P. thornei ) is one of the most important short-b...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 卓侃廖金铃林柏荣王宏洪
Owner SOUTH CHINA AGRI UNIV
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