Nucleic acid detection kit for rapidly detecting plasmodium and application of nucleic acid detection kit

A detection kit and Plasmodium detection technology, applied in the field of warm amplification, can solve the problems of undiscovered patent publications, etc., and achieve the effect of intuitive and accurate reaction results, simple operation, and easy operation

Pending Publication Date: 2022-07-29
江苏省血液中心(江苏省医学生物制品研究所)
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Through the search, no patent publications related to the application of the present invention have been found

Method used

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  • Nucleic acid detection kit for rapidly detecting plasmodium and application of nucleic acid detection kit
  • Nucleic acid detection kit for rapidly detecting plasmodium and application of nucleic acid detection kit
  • Nucleic acid detection kit for rapidly detecting plasmodium and application of nucleic acid detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026](1) Synthesis of positive standard plasmids: According to the results of literature review, the 18S rRNA of Plasmodium was selected as the target, and the related nucleic acid sequences of 4 subtypes of 18S rRNA genes were searched on NCBI (Genebank: M54897.1, AF488000.1, AB489195.1 , AB489196.1, JQ627152.1, HQ283212.1, JF681166.1, M19172.1, U03079.1, HQ283211, GQ477744.1, U07368, U83877, DQ660817, U93233.1, X1335. , X99790, KF018656.1, AB182492.1, AJ001527.1, KF219558, KF219561.1, L48987.1, L48986.1) and download. 用DNAMAN 7.0软件进行序列比对,筛出疟原虫高度保守序列如下(SEQ ID No.1):CCATTAATCAAGAACGAAAGTTAAGGGAGTGAAGACGATCAGATACCGTCGTAATCTTAACCATAAACTATGCCGACTAGGTGTTGGATGATAGTGTAAAAAATAAAAGAGACATTCTTATATATGAGTGTTTCTTTTAGATAGCTTCCTTCAGTACCTTATGAGAAATCAAAGTCTTTGGGTTCTGGGGCGAGTATTCGCGCAAGCGAGAAAGTTAAAAGAACCGACGGAAGGGGACACAGGCGTGGAGCTTGCGGCTTAATTTGACTCAACACGGGGAAACTCACTAGTTTAAGACAAGAGTAGGATTGACAGATTAATAGCTCTTTCTTGATTTCTTGGATGGTGATGCATGGCCGTTTTTAGTTCGTGAATATGATTTGTCTGGTTAATTCCGATAACGAACGAGATCTTAACCT...

Embodiment 2

[0055] Example 2 Optimization of the detection method of the Plasmodium nucleic acid detection kit (test strip method)

[0056] 1. RAA amplification

[0057] 1. Plasmodium RAA amplification system is as follows: 42.5 μL of reaction buffer, 2.5 μL of 240 mM magnesium acetate, 5 μL of negative control and template nucleic acid are added to the lyophilized powder of the reaction system prepared in Example 1. The negative control was sterilized double-distilled water.

[0058] Ⅱ. Put the reaction tube into the palm centrifuge for instant centrifugation, and centrifuge the magnesium acetate into the reaction tube;

[0059] Ⅲ. Gently flick the reaction tube to mix the reagents, and then place the reaction tube in a palm centrifuge for instant centrifugation;

[0060] Ⅳ. Place the reaction tube in a constant temperature water bath at 37°C for 4 minutes;

[0061] Ⅴ. After the time is over, gently flick the reaction tube to mix the reagents, and then place the reaction tube in a pal...

Embodiment 3

[0067] Example 3 Plasmid sensitivity, genome sensitivity and specificity detection experiment of Plasmodium nucleic acid detection kit (test strip method) detection method

[0068] 1. Plasmid Sensitivity Experiment

[0069] Use the concentration of 1copy / μL, 10copies / μL, 10 2 copies / μL and 10 3 copies / μL plasmid was used as a template to evaluate the established Plasmodium RAA-lateral flow chromatography test strip method, and 37° C. was selected to react for 20 min. The specific operation steps were the same as those in Example 2.

[0070] The results showed that the plasmid sensitivity of the Plasmodium nucleic acid detection kit (test strip method) was 1copy / μL ( image 3 ).

[0071] 2. Genome Sensitivity

[0072] Ⅰ Determination of genomic nucleic acid concentration of 4 subtypes of Plasmodium

[0073] Using Nano 2000 for genome concentration determination, the four nucleic acid concentrations were: 25.94ng / μL for Plasmodium ovale, 14.30ng / μL for Plasmodium vivax, 23.71n...

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Abstract

The invention discloses a nucleic acid detection kit for rapidly detecting plasmodium and application of the nucleic acid detection kit. The kit comprises a plasmodium upstream primer, a plasmodium downstream primer and a probe, and nucleotide sequences of the upstream primer and the downstream primer are respectively shown as SEQ ID No.2 and SEQ ID No.3 in a sequence table; the nucleotide sequence of the probe is shown as SEQ ID No.4 in a modified sequence table. The kit can rapidly and sensitively detect plasmodium, does not need expensive instruments and equipment, is simple to operate, easy in observation of reaction results and good in specificity, is very suitable for field screening of high-risk population and plasmodium prevalence areas with underdeveloped economic conditions, and is easy to popularize and use in a large range.

Description

technical field [0001] The invention relates to a nucleic acid isothermal amplification technology, in particular to a nucleic acid detection kit for rapid detection of Plasmodium and its application. Background technique [0002] Plasmodium detection methods include direct use of microscopy to find malaria parasites. The sensitivity of the most experienced microscopy technicians is protozoa (5-50) / mL; the sensitivity of routine laboratory microscopy is 500 Plasmodium / mL. Negative test takes at least 20 minutes. It is suitable for people with symptoms of infection. It cannot be used for routine screening in blood stations, nor for asymptomatic blood donors who are infected with low-titer Plasmodium. Blood stations are not equipped with experienced microscopy technicians. Cope with hundreds to thousands of specimens per day. Antibody screening is currently the most common method for selective screening of blood donors. Excluding antibody-positive donors from donating blood i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6893C12Q1/6844C12Q1/6804C12N15/11C12R1/90
CPCC12Q1/6893C12Q1/6844C12Q1/6804C12Q2521/507C12Q2531/119C12Q2565/625C12Q2563/131
Inventor 林红赵松刘艳红应清介杨坤邵雷
Owner 江苏省血液中心(江苏省医学生物制品研究所)
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