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Primer, probe, kit and method for visually and rapidly detecting nucleic acid of schistosoma japonicum katsurada through LFD-RPA

A schistosome nucleic acid and kit technology, applied in the field of LFD-RPA primers for visual and rapid detection of Schistosoma japonicum nucleic acid, achieves the effects of wide reaction temperature range, fast response and simple operation

Pending Publication Date: 2021-06-25
中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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Problems solved by technology

In addition, its amplification products can be detected by agarose gel electrophoresis, and can also be visualized by combining with lateral flow chromatography test strips, nucleic acid dyes and other methods, but nucleic acid dyes have inhibitory effect on nucleic acid amplification

Method used

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  • Primer, probe, kit and method for visually and rapidly detecting nucleic acid of schistosoma japonicum katsurada through LFD-RPA
  • Primer, probe, kit and method for visually and rapidly detecting nucleic acid of schistosoma japonicum katsurada through LFD-RPA
  • Primer, probe, kit and method for visually and rapidly detecting nucleic acid of schistosoma japonicum katsurada through LFD-RPA

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1: the preparation of template DNA, primer and probe

[0045] 1) Template DNA preparation: Tissue DNA extraction kit ( Blood&Tissue kit (purchased from Qiagen)) was used to extract genomic DNA from Schistosoma japonicum adults, Schistosoma mansoni adults, Schistosoma haematobium eggs, Paragonimus westermani metacercariae, Clonorchis sinensis adults, mixed snails, and field snails.

[0046] 2) Design of primers and probes: with the G55A gene fragment as the target sequence, RPA primers and probes were designed using Primer primer 5 software combined with BLAST, and the length of the amplified product was 235 bp. All primers and probe DNA were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0047] The upstream primer (RPA-PF) is: 5'-CCAATAAGGGCCGCTGACAGTTTATAGAGA-3';

[0048] The downstream primer (RPA-PR) is: 5'-Biotin-CAACTACCAACGGTCAGTAGCTTCATGAGC-3'

[0049] Probe (RPA-nfo probe): 5'-FAM-CACCACACACTCACCATAATGTTTGAAGAA(THF)TAATAAGTAAATATTGC-C3 S...

Embodiment 2

[0050] Example 2: Establishment and condition optimization of LFD-RPA detection method for Schistosoma japonicum genomic DNA

[0051] 1) Establishment of LFD-RPA detection method:

[0052] refer to nfo kit instructions, each 2.1 μl of the upstream and downstream primers (10 μmol / L) designed in Example 1, 0.6 μl of the probe (10 μmol / L) designed in Example 1, 29.5 μl of reaction buffer, ddH 2 O 12.2 μl, template DNA 1 μl, MgAc 2 2.5 μl was configured to form a 50 μl mixed system, added to an RPA reaction tube to dissolve the lyophilized powder, mixed well and then reacted in a water bath, using double distilled water as a negative control. The reaction temperature is set to 7 gradients: 20, 25, 30, 35, 40, 45 and 50°C, the reaction amplification time is set to 7 gradients: 0, 5, 10, 15, 20, 25 and 30min, and the other conditions are the same , to determine the optimal RPA reaction temperature and time.

[0053] RPA amplification products were detected using Milenia GenLin...

Embodiment 3

[0058] Example 3: LFD-RPA detection sensitivity and specificity evaluation of Schistosoma japonicum

[0059] Select 39°C, 20min as the RPA amplification condition, and set the Schistosoma japonicum genomic DNA template to a plasmid concentration of 10 5 、10 4 、10 3 、10 2 , 10, 1, 0.1 copy / μl and other 7 concentration gradients, and set up negative controls to evaluate the detection sensitivity of LFD-RPA.

[0060] Choose 39°C, 20min as the RPA amplification condition, set the Schistosoma japonicum genomic DNA template to 7 concentrations of 1ng / ul, 100pg / μl, 10pg / μl, 1pg / μl, 100fg / μl, 10fg / μl, 1fg / μl Gradient, set a negative control, in order to evaluate the detection sensitivity of LFD-RPA.

[0061] The specificity of LFD-RPA detection was evaluated by using 1 ng each of the genomic DNA of Schistosoma mansoni, Schistosoma haematobium, Paragonimus wescheri, Clonorchis sinensis, positive snails, negative snails, positive double umbilical snails, and negative double umbilical ...

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Abstract

The invention discloses a primer, a probe, a kit and a method for visually and rapidly detecting nucleic acid of schistosoma japonicum katsurada through LFD-RPA. The sequence of the primer is shown as SEQ ID NO.1-2, and the sequence of the probe is shown as SEQ ID NO.3. The RPA primer and the probe are designed by taking a schistosoma japonicum G55A (SjG55A) gene as a target sequence, the sensitive and rapid visual detection of schistosoma japonicum nucleic acid is realized by adopting an RPA amplification technology in combination with a lateral flow chromatography test strip method, the detection limit of schistosoma japonicum genome DNA can reach 10fg, and the identification detection of schistosoma japonicum and schistosoma mansoni is expected to be realized. One positive oncomelania in 1500 negative oncomelania can be detected, operation is simple and rapid, no special instrument is needed, the reaction temperature is close to the room temperature, the result can be observed by naked eyes, and detection and monitoring of the schistosome intermediate host in a laboratory and on site are facilitated.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a primer, a probe, a reaction system, a kit and a detection method for visual and rapid detection of Schistosoma japonicum nucleic acid by LFD-RPA. Background technique [0002] Schistosomiasis is an ancient helminth disease mainly distributed in tropical and subtropical regions. It is estimated that schistosomiasis is currently prevalent in 78 countries and regions around the world, about 800 million people are at risk of infection, 440 million people are infected with schistosomiasis, and there are about 280,000 deaths every year, which is extremely harmful to humans. The schistosomes that infect the human body mainly include S. japonicum, S. mansoni, S. mekongi, S. intercalatum, S. haematobium and horses. There are 6 kinds of schistosomiasis (S.malayensis). Only schistosomiasis is endemic in my country. After 70 years of prevention and control, my country has m...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2521/507C12Q2522/101C12Q2565/625C12Q2521/319C12Q2563/131
Inventor 邓王平许静王丽萍吕超秦志强冯婷李石柱周晓农
Owner 中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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