Primers, probe, kit and method for RPA-LFD visualization rapid detection of Schistosoma nucleic acid

A technology of schistosomiasis nucleic acid and kit, which is applied in the field of primers for RPA-LFD visualization and rapid detection of schistosomiasis circulating nucleic acid, which can solve the problems of insufficient sensitivity, limited popularization and application, and unsuitability for grassroots applications

Pending Publication Date: 2020-10-16
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 +1
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Problems solved by technology

However, most of the reported RPA or RAA-based nucleic acid detection methods for Schistosoma japonicum (SjR2) fragments target the Schistosoma japonicum non-long terminal repeat retrotransposons (SjR2) fragments. The sensitivity is good, but the sensitivity is not ideal, and the corresponding equipment is needed, so it is not su...

Method used

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  • Primers, probe, kit and method for RPA-LFD visualization rapid detection of Schistosoma nucleic acid
  • Primers, probe, kit and method for RPA-LFD visualization rapid detection of Schistosoma nucleic acid
  • Primers, probe, kit and method for RPA-LFD visualization rapid detection of Schistosoma nucleic acid

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Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: the preparation of template DNA, primer and probe

[0040] 1) Template DNA preparation: Tissue DNA extraction kit ( Blood&Tissue kit (purchased from Qiagen company) to extract Schistosoma japonicum adult worms, Schistosoma mansoni adult worms, Schistosoma haematobium ova, Paragonimus wescheri metacercariae, and Clonorchis sinensis adult worms (the five parasites were all provided by the Chinese Center for Disease Control and Prevention. Genomic DNA provided by CDC). will be 10 -2 ng, 10 -1 ng, 1ng, 10ng, and 10 2 ng Genomic DNA of Schistosoma japonicum adult worms were mixed into 500 μl of normal mouse serum, and E.Z.N.A. TM The Circulating DNA Kit was used to extract free DNA from mock-positive serum, sera of mice before and 7, 21 and 35 days after infection with Schistosoma japonicum.

[0041] 2) Design of primers and probes: With the SjCHGCS19 gene fragment as the target sequence, RPA primers and probes were designed using Primer primer 5 softwar...

Embodiment 2

[0046] Example 2: Establishment and condition optimization of the method for detecting Schistosoma japonicum genomic DNA by RPA-LFD

[0047] 1) Establishment of RPA-LFD detection method:

[0048] 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 8 gradients: 15, 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, others The conditions are the same to determine the optimal RPA reaction temperature and time.

[0049] RPA amplification products were detected using Milenia ...

Embodiment 3

[0054] Example 3: RPA-LFD detection sensitivity and specificity evaluation of Schistosoma japonicum

[0055] Select 39°C, 20min as the RPA amplification condition, and set the Schistosoma japonicum genomic DNA template to 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 ng and other 8 concentration gradients to evaluate the detection sensitivity of RPA-LFD.

[0056] The specificity of RPA-LFD detection was evaluated with 1 ng each of the genomic DNA of Schistosoma mansoni, Schistosoma haematobium, Paragonimus westermani and Clonorchis sinensis as templates.

[0057] The results showed that: except double distilled water and 10 -7 ng group outside, 10 -1 ~10 -6 In the ng group, obvious detection line bands could be seen, and with the increase of the template amount, the color of the detection line gradually deepened ( figure 2 A), the minimum detection limit of RPA-LFD to Schistosoma japonicum genomic DNA can reach 10 -6 ng(1fg). The results of the specificity evalu...

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Abstract

The invention discloses primers, probe, kit and method for the RPA-LFD visualization rapid detection of Schistosoma nucleic acid. Sequences of the primers are as shown in SEQ ID NO.1-2, and a sequenceof the probe is as shown in SEQ ID NO.3. The RPA primers and probe are designed with the SjCHGCS19 gene as a target sequence, and the sensitive and rapid visualization detection of the schistosome nucleic acid is realized by using a RPA amplification technology combined with lateral flow chromatography test strip method. The detection method of the invention has a detection limit of 1 fg for Schistosoma japonicum genome DNA, and is expected to be used in the general detection of Schistosoma mansoni and Schistosoma haematobium. The method can detect circulating nucleic acid of Schistosoma in the serum of mice at an early stage of infection, the operation is simple and fast, no special equipment is required, a reaction temperature is close to room temperature, results can be observed with the naked eye, and the realization of the early and sensitive and rapid detection of intermediate hosts of Schistosoma on site, and the timely and sensitive monitoring of environments with high schistosomiasis transmission risks 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 RPA-LFD visual rapid detection of schistosome circulating nucleic acid. Background technique [0002] Schistosomiasis is a serious zoonotic parasitic disease that is prevalent in 78 countries and regions around the world, and is listed by the WHO as one of the neglected tropical diseases that are easily recurring. Only schistosomiasis is prevalent in China. After 70 years of active prevention and control, the infection rate and degree of schistosomiasis in my country have been reduced to an extremely low level in history. At the same time, with the acceleration of the global integration process, the flow of people has become more frequent, and imported cases of schistosomiasis continue to appear, which has become a new challenge and new difficulty for the prevention and control of schistosomiasis ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6893C12Q1/6844C12N15/11
CPCC12Q1/6893C12Q1/6844C12Q2521/507C12Q2522/101C12Q2565/625
Inventor 周晓农胡薇邓王平洪清华许静徐斌李石柱
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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