LAMP primer group, kit and method for rapid identification of tsutsugamushi disease rickettsia

A Rickettsia and RT-LAMP technology, applied in the field of detection and identification of pathogenic microorganisms, can solve the problems of poor sensitivity, expensive equipment, difficulties, etc., and achieve the effect of good specificity, high sensitivity and high specificity

Inactive Publication Date: 2015-10-21
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it is difficult to detect the pathogen culture of scrub typhus, and conventional serological typing has the disadvantages of long detection time, poor specificity, and poor sensitivity.
Nucleic acid detection methods such as fluorescent PCR, although the sensitivity and specificity of detection have been improved, but expensive instruments and equipment are required
Although the above various methods may provide better diagnostic value clinically, they cannot be used on a large scale clinically due to their respective shortcomings.
Loop-Mediated Isothermal Amplification Technology (Loop-Mediated Isothermal Amplification, LAMP) is a gene amplification technology developed by Eiken Chemical Co., Ltd. in Japan around 2000. It has the advantages of fast and simple, accurate operation, easy popularization, safety and reliability However, the LAMP method has not yet been applied to the rapid identification of Rickettsia tsutsugamushi

Method used

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  • LAMP primer group, kit and method for rapid identification of tsutsugamushi disease rickettsia
  • LAMP primer group, kit and method for rapid identification of tsutsugamushi disease rickettsia
  • LAMP primer group, kit and method for rapid identification of tsutsugamushi disease rickettsia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Establishment of RT-LAMP kit for rapid identification of Rickettsia tsutsugamushi

[0032] RT-LAMP kit for rapid identification of Rickettsia tsutsugamushi, including the following components: (1) specific primer set; (2) reverse transcriptase; (3) DNA polymerase; (4) RT-LAMP reaction solution; (5) chromogenic reagent; (6) positive control and negative control.

[0033] (1) Design of specific primers:

[0034] Six specific primers were designed according to the specific region of Rickettsia tsutsugamushi (GenBank accession number: GU446621.1), and the sequences were as follows:

[0035] Internal primer 1: 5'-TTGCTACACCAAGTGCTCCTGATATGCTGGTCTTGGTGC-3' (SEQ ID No.1);

[0036] Internal primer 2: 5'- TTAATGCTGCTGAGGGTGTGTCGCATTTACCGAGTACTTATCT-3' (SEQ ID No.2);

[0037] Outer primer 1: 5'-TTGATCTGAGTATGATTGTCGG-3' (SEQ ID No.3);

[0038] Outer primer 2: 5'-GAAGTTATAGCGTACACCTACA-3' (SEQ ID No.4);

[0039] Loop primer 1: 5'-GCAACCATACCTGTATGCC-3' (SEQ ID No....

Embodiment 2

[0047] Example 2 Detection method for Rickettsia tsutsugamushi

[0048] (1) Extract template RNA.

[0049] (2) Using the specific primer set in Example 1 to perform a loop-mediated isothermal gene amplification reaction on the template RNA:

[0050] The 25 μL reaction system contains: the final concentration of inner primer 1 and 2 is 8 pmol / μL, the final concentration of outer primer 1 and 2 is 1 pmol / μL, the final concentration of loop primer 1 and 2 is 4 pmol / μL, and the reaction solution is 12.5 μL , 8 U of DNA polymerase, 50 ng of RNA to be tested, 1 U of reverse transcriptase, made up to 25 μL with sterilized deionized water; the conditions of the loop-mediated isothermal gene amplification reaction are: 63 ° C for 40 min.

[0051] (3) Judgment of results: Add 2 μL of chromogen 10×SYBR GREEN I to the above reaction tube, and observe the color of the reaction solution after mixing. If it is green, it is Rickettsia tsutsugamushi, and if it is orange, it is Rickettsia ts...

Embodiment 3

[0052] Embodiment 3 specificity experiment

[0053] Utilize the method of embodiment 2 to respectively treat 2 kinds of herpes virus (HSV, EBV), Japanese encephalitis virus (JEV), yellow fever virus (YFV), 2 types of dengue fever virus (DENV2), rickettsia pratzii ( Rickettsia mooseri), Rickettsia mooseri, and Rickettsia mooseri were detected, and DEPC water was used as a negative control.

[0054] See the test results image 3 . Only the Rickettsia tsutsugamushi tubes are green, the rest of the tubes are orange. The results show that the detection kit of the present invention has high specificity, and can accurately detect Rickettsia tsutsugamushi and Rickettsia prauszii, Rickettsia moschii, Rickettsia rickettsia and other non-related viruses differentiate( image 3 ).

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Abstract

The invention discloses an LAMP primer group, kit and method for rapid identification of tsutsugamushi disease rickettsia. The detection primer group comprises a pair of outer primers, a pair of inner primers and a pair of loop primers. The detection kit comprises primer liquid, reaction liquid, DNA polymerase, reverse transcriptase and a contrasting and color developing agent. The detection method comprises the steps that by extracting the pathogene RNA to be detected, amplification is conducted on a sample RNA template at the temperature of 60-65 DEG C by means of the reverse transcription activity of the reverse transcriptase through six specific primers and a DNA polymerase having the strand displacement activity, and whether amplification is achieved or not is judged by adding the color developing agent and observing the change of color in a reaction tube. The LAMP primer group, kit and method for rapid identification of the tsutsugamushi disease rickettsia have the advantages of being rapid, efficient, easy and convenient to operate, high in specificity, high in sensitivity, capable of achieving identification easily and conveniently, suitable for field detection and the like, and the LAMP primer group, kit and method for rapid identification of the tsutsugamushi disease rickettsia are suitable for application and popularization.

Description

technical field [0001] The invention relates to the detection and identification of pathogenic microorganisms, in particular to a method for rapidly identifying Rickettsia tsutsugamushi by using a loop-mediated isothermal gene amplification technique (LAMP technique). Background technique [0002] Scrub typhus, also known as scrub typhus, is a disease caused by Rickettsia tsutsugamushi, and is one of the earliest infectious diseases discovered in China. Rickettsia tsutsugamushi is short rod-shaped, with an average length of 1.2um, usually arranged in pairs, living in chiggers, and can be passed down through eggs. Chigger larvae need to suck the lymph fluid or blood of humans or animals to complete the development process from larvae to nymphs. After biting the body, they can cause chills, fever, eschar or ulcers on the bites, swollen lymph nodes , conjunctival hyperemia, rash and other clinical symptoms. Scrub typhus is widely prevalent in my country, and can be divided in...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12Q1/68
Inventor 黄曦刘泽霞
Owner SUN YAT SEN UNIV
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