Primer combination, and detection reagent or kit for anisakis

A combination of primers and anisakis technology, applied in the biological field, can solve problems such as contamination of false positive results, complex primer design, environmental pollution, etc., and achieve the effect of portable nucleic acid detection, simple primer design, and large development space

Active Publication Date: 2021-03-16
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the primer design of the LAMP method is complex, large in number, and difficult. It is necessary to design 4 pairs of primers according to 6 specific regions, and the amplification products are fragments of different sizes, which are easy to form aerosols and cause environmental pollution in the laboratory.
Therefore, the LAMP reaction is easily contaminated and produces false positive results, which has high requirements for operators and testing environment

Method used

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  • Primer combination, and detection reagent or kit for anisakis
  • Primer combination, and detection reagent or kit for anisakis
  • Primer combination, and detection reagent or kit for anisakis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The identification of embodiment 1 anisakis

[0048] Extraction of parasite genomes (Anisakis, Schizocephalus lacunae, Clonorchis sinensis, Mesorchis orientalis, Echinostomum)

[0049] (1) Take out the parasites from the -80°C refrigerator, freeze and thaw them repeatedly with liquid nitrogen and boiling water for 3-5 times, 30s each time.

[0050] (2) Add 500 μL of lysate to a 1.5 mL centrifuge tube, then add 20 μL of 2% proteinase K, shake and mix.

[0051] (3) Put the centrifuge tube in a 56°C water bath, incubate for 2 hours, and turn the centrifuge tube 3-5 times every hour (the purpose of turning is to fully lyse the tissue).

[0052] (4) Add 5 μL of RNaseA, mix well and let stand for 2 minutes.

[0053] (5) Add an equal volume of Tris-saturated phenol (500 μL), vortex vigorously and mix well, then let stand for 10 min.

[0054] (6) Centrifuge at 12,000rpm at 4°C for 15min. After centrifugation, it is divided into upper, middle and lower layers. The upper layer...

Embodiment 2

[0065] Example 2 Primer Design and RPA Condition Optimization

[0066] According to the sequence comparison of the ITS region of the identified insect species by DNAMAN, 8 pairs of RPA amplification primers were designed, namely ITS-1, ITS-2, ITS-3, ITS-4, ITS-5, ITS-6, ITS -7, ITS-8. The sequence is as follows:

[0067] ITS-1: F, 5'-CTAGGTGGCCGCCAAAACCCAAAACACAACC-3';

[0068] R, 5'-ACAGTTCACCGTAATTCGACCCTCAGCCAGACG-3';

[0069] ITS-2: F,5'-CTAGGTGGCCGCCAAAACCCAAAACACAACC-3';

[0070] R, 5'-ACGAACCGAGTGATCCACCGCCAAGATTTG-3';

[0071] ITS-3: F,5'-TGCGATAAATAGTGCGAATTGCAGACACAT-3';

[0072] R,5'-AATTGCTTGCCGAATGCTTCACAGTCCAGAAAAA-3';

[0073] ITS-4: F,5'-GTCAGTTGCGATGAAAGATGCGGAGAAAGTTCC-3';

[0074] R, 5'-TGCTCAATGTGTCTGCAATTCGCACTATTTATCG-3';

[0075] ITS-5: F,5'-TGTTGAACAACGGTGACCAATTTGGCGTCTACG-3';

[0076] R, 5'-AGTGATCCACCGCCAAGATTTGTACATTTTCAACACAT-3';

[0077] ITS-6: F,5'-TGTTGAACAACGGTGACCAATTTGGCGTCTACG-3';

[0078] R, 5'-AGCTGGCTGCGTTTCTTCATCGATCCACGAA-3';...

Embodiment 3

[0091] Embodiment 3 Sensitivity and specificity detection

[0092] Sensitivity test: use Nanodroop 2000 to measure the concentration of Anisakis uteri DNA (100.5ng / μL), and dilute it with sterile water 10 times to 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL. RPA reactions were performed using diluted DNA at different concentrations as templates to determine the sensitivity of the method.

[0093] Specificity detection: respectively adopt the nematode uteri (100.5ng / μL), anisakis simplex (85.7ng / μL), anisakis peizii (60.3ng / μL), paraceca nematode (53.2ng / μL), Typical Anisakis (60.2 ng / μL) DNA was used as a template, and at the same time, Plasmocephalus ladenum (236.15 ng / μL), Clonorchis sinensis (200.6 ng / μL), Mesorchis orientalis (132.7 ng / μL), Echinostoma nematode (154.6ng / μL) was used as negative control and double distilled water was used as blank control, and RPA method was used for amplification. Add 1.5 μL of the genome of each worm body.

[0094] Sensitivity and sp...

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Abstract

The invention relates to the field of biology, in particular to a primer combination, and a detection reagent or kit for anisakis. According to the invention, sequence alignment is performed through DNAMAN according to the obtained ITS regions of the five kinds of anisakis, so that a universal primer is designed. A RPA method established by the invention can amplify a target fragment of an anisakis genome at about 340bp, can specifically detect the anisakis, and has negative detection results on broad tapeworm, clonorchis sinensis, metorchis orientalis and gnathostoma spinigerum. After optimization, detection can be completed at 35 DEG C within 25 min, the sensitivity can reach 1 pg/[mu] L, and the detection result of an artificial simulation contaminated sample is positive. The method issimple and convenient to operate, and has important significance for on-site rapid detection.

Description

technical field [0001] The invention relates to the biological field, in particular to a primer combination and anisakis detection reagent or kit. Background technique [0002] Foodborne parasitic diseases refer to a general term for a class of diseases that are infected by eating raw or not thoroughly heated and cooked foods containing parasite eggs or larvae. In recent years, with the improvement of people's living standards and the diversification of dietary sources and methods, outbreaks of foodborne parasitic diseases have occurred from time to time, and food safety problems caused by food parasites have become more prominent. Foodborne parasitic diseases are not only one of the main factors affecting the health of our people, but also one of the important global disease burdens. [0003] Anisakis is an important food-borne zoonotic parasite with a wide range of distribution. Many fish and cephalopods can be infected with Anisakis. The larvae of Anisakis are mainly pa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/6888C12Q1/6844
CPCC12Q1/6844C12Q1/6888C12Q2531/119C12Q2521/507C12Q2537/1376C12Q2565/125
Inventor 刘明远刘晓雷白雪杨勇唐斌李辰张春玲
Owner JILIN UNIV
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