Haemonchus contortus nucleic acid detecting primer pair and probe and rapid detection kit

A technology of Haemonchus contortus and a detection kit, which is applied to biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., and can solve the problem of not being able to obtain the infection and prevalence of Haemonchus contortus in time, Drug resistance, unable to realize on-site detection in farms and other problems, to achieve the effect of simple operation, strong specificity and high sensitivity

Inactive Publication Date: 2018-02-02
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

However, the long-term use of anti-helminth drugs makes Haemonchus contortus drug resistance common in pastures
Timely detection of the infection and prevalence of Haemonchus contortus can play an important role in curbing the spread of Haemonchus contortus, but none of the existing detection methods can realize on-site detection of Haemonchus contortus, and cannot obtain Haemonchus contortus in time infection and prevalence of

Method used

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  • Haemonchus contortus nucleic acid detecting primer pair and probe and rapid detection kit
  • Haemonchus contortus nucleic acid detecting primer pair and probe and rapid detection kit
  • Haemonchus contortus nucleic acid detecting primer pair and probe and rapid detection kit

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Experimental program
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Effect test

Embodiment 1

[0031] The rapid detection kit for the nucleic acid of Haemonchus contortus of the present invention includes:

[0032] LF-RPA primer probe mix, RPA reaction master mix (29.5μl Rehydration Buffer, 2.5μl 280mM Magnesium Acetate mix and 5mg freeze-dried enzyme) provided by RPA kit (TwistAmp nfo kit, TwistDx), PCRD kit (PCRD) Nucleic Acid Detection, TwistDx) provides PCRD test strips and PCRDextraction buffer.

[0033] The specific preparation method is:

[0034] (1) LF-RPA primer-probe mixture: This application designs primers and probes for the ITS-2 gene of Haemonchus contortus. The specific nucleotide sequence is as follows:

[0035] Upstream primer: 5'--CAAATGGCATTTGTCTTTTAG-3';

[0036] Downstream primer: 5'-biotin-TTAGTTTCTTTTCCTCCGCTAAATGATATG-3';

[0037] Probe: 5'-FAM-TATTGAGATTGACTTAGATAGTGACTTGTA-THF-GGCGACGATGTTCTT-SpacerC3-3'.

[0038] Mix the upstream primer 23poml, the downstream primer 23pmol, and the probe 2pmol in 16μl of pure water to obtain the primer-probe mixture and ...

Embodiment 2

[0044] Specific application examples:

[0045] The method for rapidly detecting the nucleic acid of Haemonchus contortus using the kit of the present invention is as follows:

[0046] (1) RPA amplification reaction:

[0047] Take 2μl of nucleic acid sample, add 16μl of primer probe mixture and 32μl of RPA reaction premix into the reaction tube and mix. React for 15-20 minutes at 30°C-45°C (you can put it in your hands or under your armpit).

[0048] (3) Detection of reaction products:

[0049] After the reaction, draw 1μl of reaction product and 79μl PCRD extraction buffer and mix them, and add all of them to the loading hole of PCRD test strip. Observe the result after 10 minutes at room temperature. One bar is negative and two bars are positive.

Embodiment 3

[0051] Sensitivity test of the kit of the present invention:

[0052] The experiment process is: after extracting DNA from pure Haemonchus contortus stage 3 larvae, measuring the DNA content with an ultraviolet spectrophotometer, and performing a gradient dilution of the DNA; performing an RPA amplification reaction on the gradient dilution of the DNA, each amplified The amount of Haemonchus contortus DNA added in the amplification reaction was 10pg, 1pg, 100fg, 10fg, 1fg and the blank control in sequence. After the reaction, PCRD test strip detection was performed according to the aforementioned method. The results showed that the kit can detect at least 10 fg of Haemonchus contortus DNA in the reaction system.

[0053] See experimental results figure 1 (Note: The appearance of band C on the PCRD test strip indicates that the test strip functions normally, and the appearance of 2 bands indicates positive).

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Abstract

The invention discloses a haemonchus contortus nucleic acid detecting primer pair and probe. Nucleotide sequences of upstream and downstream primers of the primer pair are shown as a sequence 1 and asequence 2 in a sequence table, and a nucleotide sequence of the probe is shown as a sequence 3 in the sequence table. The invention further discloses a rapid detection kit and a detection method. Thehaemonchus contortus nucleic acid detecting primer pair and probe and the rapid detection kit are established on the basis of molecular biology, the primer pair and the probe are capable of realizingspecific amplification of haemonchus contortus, the rapid detection kit for rapid haemonchus contortus nucleic acid detection is higher in sensitivity than PCR (polymerase chain reaction) and is highin specificity and detection speed, and high simplicity and convenience in result determination operation are achieved. By combination of the detection method, the detection kit and an existing nucleic acid rapid extraction method, farm onsite detection in a non-lab environment can be realized, and accordingly measures can be taken timely to clear haemonchus contortus.

Description

Technical field [0001] The invention relates to the technical field of parasite detection, in particular to a primer pair and a probe for detecting the nucleic acid of Haemonchus contortus and a rapid detection kit. Background technique [0002] Haemonchus contortus is an important parasite that infects ruminants in Trichostrongylidae. The females of Haemonchus contortus lay a high amount of eggs, and can lay 5000-10000 eggs per day. The temperature, humidity, oxygen, and light of the grassland are all conducive to the eggs hatching into invasive larvae, and the temperature in winter cannot freeze to death. Overwintering insect eggs on the grassland often cause serious pollution of the grassland and cause serious economic losses to the livestock industry, especially the sheep industry. Adult worms mainly parasitize on the true gastric mucosa of sheep and suck blood. After infection, the clinical symptoms are anemia, diarrhea and edema, etc., and even death. The mortality rate ca...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2521/507C12Q2522/101C12Q2565/625
Inventor 吴耀东周东辉王琪琪邹扬胡敏徐民俊朱兴全
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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