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Rapid detection method for trichina nucleic acid and rapid detection kit thereof

A detection kit and detection method technology, applied in the biological field, can solve the problem of no effective prevention method being found, and achieve the effects of simple operation, strong specificity and high sensitivity

Pending Publication Date: 2017-11-24
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, no effective prevention method has been found for the disease. The main prevention and control measures are to strictly inspect the pollution of meat products and encourage people not to eat raw meat foods. Methods of preventing infection in humans

Method used

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  • Rapid detection method for trichina nucleic acid and rapid detection kit thereof
  • Rapid detection method for trichina nucleic acid and rapid detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Trichinella spiralis nucleic acid rapid detection kit of the present invention comprises:

[0030] RPA primer-probe mixture, PBST solution, flow chromatography test strips (MileniaHybridtech 1strips, Milenia Biotec), lyophilized enzyme provided by RPA kit (TwistAmp nfo kit, TwistDx) and RPA reaction premix (29.5 μL Rehydration Buffer and 2.5μL 280mM Magnesium Acetate mixture).

[0031] The specific preparation method is:

[0032] (1) Primer-probe mixture: This application is aimed at rrnS gene primers and probes of Trichinella spiralis, and the specific nucleotide sequence is as follows:

[0033] Upstream primer: 5′-CATGGTTAGGTGAGATATTGCCTGCAAATA-3′;

[0034] Downstream primer: 5′-biotin-GGTCCTCCTTTCCAGAAGATCTACTTTGTTA-3′;

[0035] Probe:

[0036] 5'-FAM-CCCACTAAATTCCTTATGCACATATTGCCC-dSpacer-TCACCCTCATAAGAG-C3Spacer-3';.

[0037] 21 poml of the upstream primer, 21 pmol of the downstream primer, and 6 pmol of the probe were mixed in 16 μL of pure water to obtain a ...

Embodiment 2

[0043] Specific application examples:

[0044] The method for rapidly detecting Trichinella spiralis using the kit of the present invention is as follows:

[0045] (1) RPA amplification reaction:

[0046] Pipette 2 μL of nucleic acid sample, add 16 μL of prepared primer-probe mixture and 32 μL of RPA reaction master mix into the centrifuge tube containing RPA reaction lyophilized enzyme, and mix well. React for 5-25 minutes under the condition of 25°C-45°C (can be held in hands or under armpits).

[0047] (3) Detection of reaction products:

[0048] After the reaction, draw 2 μL of RPA amplification product and mix with 198 μL PBST solution, then draw 10 μL of the mixed solution and add it dropwise to the sample loading end of the flow chromatography test strip, and finally insert the test strip vertically into the tube containing 200 μL of PBST solution. In the centrifuge tube, observe the results after 5 minutes. If the test strip shows two bars, it is positive for Trichi...

Embodiment 3

[0050] The sensitivity experiment of kit of the present invention:

[0051] The experimental process is as follows: DNA is extracted from purified Trichinella spiralis, and RPA amplification reaction is performed, and the amount of Trichinella spiralis DNA added in each amplification reaction is 1ng, 100pg, 10pg, 1pg, 100fg, 10fg respectively. After the reaction, flow chromatography test strip detection was carried out according to the aforementioned method, and the results showed that this method could detect 100 fg of Trichinella spiralis DNA added to the reaction system.

[0052] For experimental results, see figure 1 (Note: the appearance of the control band is to show that the function of the test strip is normal, and the appearance of the detection band indicates positive; NC is a blank control).

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Abstract

The invention discloses a rapid detection kit for trichina nucleic acid. The kit comprises the following components: an RPA primer probe mixed solution, a PBST solution, a flow measurement chromatographic test strip, RPA lyophozyme and an RPA reaction premixed solution. The invention also provides a rapid detection method for the trichina nucleic acid. The rapid detection method and the rapid detection kit have the beneficial effects that on the basis of molecular biology, a specific primer and a probe are firstly designed on the basis of trichina mitochondrion small subunit rRNA (rrnS) genes, then the kit for rapidly detecting the trichina nucleic acid is prepared by utilizing a recombinant enzyme polymeric enzyme amplification-flow measurement chromatography method, the sensitivity is higher than that of traditional PCR, the specificity is high, a whole process only needs 30 minutes, and a result is extremely simple and convenient to determine; and after a nucleic acid sample is obtained, the whole reaction process can be carried out in a non-laboratory environment, and the rapid detection method and the rapid detection kit not only are suitable for clinical diagnosis, but also can be applied to various fields such as food safety detection, farm on-site detection and wild animal environmental assessment.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a rapid detection method of Trichinella spiralis nucleic acid and a rapid detection kit thereof. Background technique [0002] Trichinella spiralis is the largest intracellular parasitic nematode that infects almost all mammals, some birds, and reptiles. It can complete the entire life cycle in the same host without the need for an intermediate host. Trichinellosis is a very important public health problem, which not only poses a serious threat to human health, but also causes serious economic losses to animal husbandry production, meat industry, and foreign trade exports. People are mainly infected by eating raw or semi-raw pork or other animal meat containing cysts of Trichinella spiralis muscle larvae, and can cause death in severe cases. At present, no effective prevention method has been found for the disease. The main prevention and control measures are to strictly inspect th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888
Inventor 付宝权李婷婷张念章李文卉
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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