Lyme disease spirochaete detection RPA primer and probe and detection method thereof

A Lyme disease helix and probe technology, applied in the fields of biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of expensive, complicated and time-consuming WB operation, and achieve the elimination of equipment investment and convenience. The effect of grassroots promotion, high specificity and sensitivity

Inactive Publication Date: 2016-04-27
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
At present, RPA technology is mainly used in the rapid detection of pathogenic microorganisms, including viruses, bacteria, fungi, mycoplasma, parasites, etc., and is widely used in clinical disease diagnosis, food hygiene inspection and environmental monitoring. Related reports on detection of spirochetes

Method used

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  • Lyme disease spirochaete detection RPA primer and probe and detection method thereof
  • Lyme disease spirochaete detection RPA primer and probe and detection method thereof
  • Lyme disease spirochaete detection RPA primer and probe and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 is used for detecting the synthesis of the RPA primer of Lyme disease spirochete and probe

[0034] The Lyme spirochete strain B31 was used for the screening of RPA primers and probes for the detection of Lyme spirochete. Taking the recA gene as the target gene, according to the requirements of the TwistDX operation manual, use Primer5 to design 5 pairs of primers that can be used in the BasicRPA kit. The 5 pairs of primers were screened using the BasicRPA kit to obtain a pair of primers that could stably amplify the target product and had the best sensitivity and specificity. See SeqIDNo:1 and 2 for the best primer sequences obtained by screening. Then, on the basis of this primer, the above primers were modified according to the requirements of the TwistDX company RPAnfo kit operation manual, and a biotin labeling site was added to the 5' end of the reverse primer. In addition, a 46-52bp primer was designed according to the RPA primer sequence The 5' en...

Embodiment 2

[0039] Example 2 Utilize RPA primers and probes to detect the specificity analysis of Lyme disease spirochete

[0040] 1.1 Reagents and equipment

[0041] A small constant temperature shaker, TwistnfoRPA kit was purchased from TwistDX Company in the UK (product number: TANFO02KIT).

[0042] 1.2 Sample source

[0043] The 36 kinds of Lyme spirochete bacterial strains adopted in the present embodiment are provided by the Chinese Center for Disease Control and Prevention, Infectious Disease Prevention and Control (Table 1); Anaplasma, Coxella beinii and Leptospira were provided by the Anaplasma Ward and Leptospira Room of the Institute of Infectious Diseases, Chinese Center for Disease Control and Prevention; Escherichia coli BL21 was purchased from Kangwei Century Biotechnology Co., Ltd. .

[0044] Table 1 Background information on Borrelia Lyme disease for RPA detection

[0045]

[0046]

[0047] 1.3 DNA extraction

[0048] DNA extraction of Borrelia Lyme disease str...

Embodiment 3

[0055] Example 3 Utilize RPA primers and probes to detect the sensitivity analysis of Lyme disease spirochete

[0056] Using Borrelia Lyme disease B31 as a template, the RPA primers LF-F and LF-R of Example 1 were used for amplification. Among them, the B31 genomic DNA uses ddH 2 O was serially diluted to 10pg / μL, 1pg / μL, 100fg / μL, 50fg / μL, 10fg / μL. Perform RPA reactions in RPA reaction tubes.

[0057] Reaction system (50μl):

[0058]

[0059] The RPA reaction conditions were: 37°C, 20 minutes, and the reaction was terminated on ice.

[0060] The amplified product was mixed with the buffer in the Hybriddetect2T kit (containing lateral flow chromatography test strips, purchased from MileniaBiotec, Germany, product number: MILENIA01) at a volume ratio of 1:20, and then the Hybriddetect2T test strips were placed in In the above mixture, read the result after 5 minutes. figure 2 It is the sensitivity detection result of RPA method. Depend on figure 2 It can be seen tha...

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Abstract

The present invention provides a lyme disease spirochaete detection RPA primer and probe, the RPA primer is designed on the basis of lyme disease spirochaete recA gene, and primer sequences are as shown in Seq ID No: 1 and 2. The probe is designed on the basis of the RPA primer, and has a size of 46-52bp, 5 'end of the probe is marked with FAM, 3' end of the probe is modified by C3Spacer, and dSpacer or THF modification is performed at a position 30bp apart from the 5 'end of the probe. The present invention further provides a kit comprising the RPA primer and the probe. The present invention further provides a lyme disease spirochaete detection RPA method baed on the RPA primer and the probe. By combined use of RPA primer isothermal amplification technology and lateral flow chromatography test paper, lyme disease spirochaetes can be quickly and accurately detected from serum of suspicious patients. The specificity and sensitivity of the method are higher than that of conventional PCR, the method can be used for detecting lyme disease spirochaetes of different pathogenic genotypes, has important significance for lyme disease early diagnosis and treatment and other aspects of the lyme disease, can eliminates excessively high instrument cost, and is easy to promote and use in grassroots.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to an RPA primer, a probe and a detection method for detecting Lyme spirochete. Background technique [0002] Lyme disease is a chronic natural foci disease caused by Borrelia burgdorferi. 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 treated early, it can often be cured, otherwise serious complications...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2521/507C12Q2521/101C12Q2522/101C12Q2531/119Y02A50/30
Inventor 郝琴刘炜张琳侯学霞刘慧鑫万康林
Owner ICDC CHINA CDC
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