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Loop-mediated isothermal amplification method for detecting lyme disease spirochete

A Lyme disease helical and loop-mediated isothermal technology, applied in the field of warm amplification method, can solve the problems of complicated, expensive, and time-consuming WB operations, and achieve the convenience of popularization and use at the grassroots level, the exemption of equipment investment, high specificity and sensitivity Effect

Active Publication Date: 2013-11-27
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

WB can judge and verify the true and false positives of IFA and ELISA, but because Lyme spirochete has different pathogenic genotypes, and the distribution of these genotypes in different parts of the world is different, so each country should according to the actual situation, Formulate WB positive diagnostic criteria for genotypes that are popular in the country, and the operation of WB is relatively complicated, which is not conducive to popularization
The disadvantages of PCR technology are: (1) the target gene is easily contaminated; (2) non-specific amplification is prone to occur; (3) the amplification reaction is easily affected by various factors; (4) it needs to invest in expensive precision Instruments (such as PCR instrument, gel electrophoresis instrument and gel imaging system); (5) It takes a long time, requiring 2 to 3 hours of reaction time and 1 to 2 hours of electrophoresis time
However, there is no report on the detection of Lyme spirochete using LAMP technology

Method used

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  • Loop-mediated isothermal amplification method for detecting lyme disease spirochete
  • Loop-mediated isothermal amplification method for detecting lyme disease spirochete
  • Loop-mediated isothermal amplification method for detecting lyme disease spirochete

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 is used to detect the synthesis of the LAMP primer set of Lyme disease spirochete

[0028] Five Lyme spirochete strains, B31, PD91, Fujiji, FP1, and QX-S13, were used to screen LAMP primers for detection of Lyme spirochete. B31 and Fuji were purchased from ATCC in the United States, and PD91, FP1 and QX-S13 were isolated, cultivated and preserved by the Institute of Infectious Diseases, China Center for Disease Control and Prevention. Using the Japanese Eiken loopamp DNA amplification kit (product number: 18001), select gene loci (fla, 16SrDNA, rrf-rrl spacer, hbb, recA, ospA, ospC, groEL) commonly used in PCR amplification reactions, and use PrimerExplorer V4 online software designed LAMP primers and synthesized primers with good parameters. The cultured B31, PD91, Fuji, FP1, and QX-S13 Lyme spirochete strains were used to screen the primers. Under the same conditions, the primers that could make these five strains reach positive and peak height as soon as p...

Embodiment 2

[0036] Example 2 Specificity analysis of detection of Lyme disease spirochete by using LAMP primer set

[0037] 1.1 Reagents and equipment:

[0038] The water bath and the LAMP kit were purchased from Eiken Corporation, Japan.

[0039] 1.2 Sample source:

[0040] 5 strains of Lyme spirochetes, 8 strains of the genus Rickettsia and Leptospira, Brucella, and Escherichia coli (Table 1) used in this example are preserved in the Institute of Infectious Diseases, China Center for Disease Control and Prevention Or the Institute of Infectious Diseases Prevention and Control, Chinese Center for Disease Control and Prevention.

[0041] Table 1 Sample sources used for LAMP detection

[0042]

[0043] 1.3 DNA extraction:

[0044] DNA extraction of Borrelia Lyme disease strains by pyrolysis: Inoculate an appropriate amount of strains into BSKII medium, culture at 33°C for 7 days, centrifuge the cultured strains at 12,000rpm for 30 minutes to collect the bacteria, remove the supernat...

Embodiment 3

[0051] Example 3 Sensitivity analysis of detection of Lyme disease spirochete by using LAMP primer set

[0052] The fla gene of PD91 was amplified by PCR using LAMP outer primers F3 and B3 as primers, and the PCR product was recovered and purified using a gel extraction kit (product number: D2500-01) from OMEGA Company. Then use the PMD-18T Vector kit (product number: D102A) of Takara Company to connect the purified DNA to the PMD-18T Vector, connect in a water bath at 16°C for 4 hours, and then transform into Escherichia coli competent cells (purchased from Tiangen Biochemical Technology Co., Ltd. (Beijing) Co., Ltd., referred to as Tiangen Company). Then Escherichia coli was inoculated into LB solid medium containing ampicillin (0.1mg / ml), cultivated overnight at 37°C, and the individual colonies that grew were respectively expanded and cultivated, and then the plasmid extraction kit (commodity batch number : J9110) to extract the plasmid and carry out clone identification ...

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Abstract

The invention provides a loop-mediated isothermal amplification method for detecting lyme disease spirochete. DNA (deoxyribonucleic acid) of a sample to be detected is subjected to constant temperature amplification reaction by adopting a FIP (forward inner primer) and a BIP (backward inner primer) and F3 and B3 (shown in Seq ID No.1-4). According to the loop-mediated isothermal amplification method, a LAMP (loop-mediated isothermal amplification) primer constant-temperature amplification technique is used for fast detecting the lyme disease spirochete, and the lyme disease spirochete can be accurately detected from suspected patient serum. The specificity and sensitivity of the method are higher than those of the common PCR (polymerase chain reaction). Different virulence genes of lyme disease spirochetes can be detected, and the method has significance in the aspects of early diagnosis and early treatment of the lyme disease and the like. Investment on an expensive apparatus can be avoided and the method is convenient for grass-root popularization and use.

Description

technical field [0001] The present invention relates to the detection of Lyme spirochetes, in particular to a loop-mediated isothermal amplification method for the detection of Lyme spirochetes. Background technique [0002] Lyme disease is a chronic natural foci disease caused by Borrelia burgdorferi. Borrelia burgdorferi is a unicellular Gram-negative spirochete. The disease is mainly transmitted from host animal to host animal and humans through the bite of arthropod ticks. In the early clinical manifestations of Lyme disease, there are typical skin lesions - chronic migratory erythema (ECM), accompanied by headache, fever, chills, fatigue and discomfort, local lymph node enlargement and other symptoms, and later manifested as nervous system, circulatory system, Various damages to the motor system that appear intermittently and alternately. It has the characteristics of wide distribution, long course of disease and high mortality rate. If it can be diagnosed and treat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCY02A50/30
Inventor 郝琴张刘丽侯学霞耿震
Owner ICDC CHINA CDC
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