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Nested fluorescent quantitative PCR (Polymerase Chain Reaction) detection method for Lyme disease spirochetes

A Lyme disease helix and detection reagent technology, which is applied in the field of molecular biology and microbial detection, can solve the problems of low sensitivity and cannot be used to determine the type of infected strains in positive specimens, so as to improve sensitivity and specificity Effect

Pending Publication Date: 2021-10-19
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the above methods, the ordinary PCR method has low sensitivity and is generally used for strain identification, and is rarely used for clinical specimen detection; the detection limit of real-time PCR for genes such as recA and hbb can only reach 10 1 -10 2 / mL, but these target genes cannot be used to determine the infection strain type of positive samples

Method used

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  • Nested fluorescent quantitative PCR (Polymerase Chain Reaction) detection method for Lyme disease spirochetes
  • Nested fluorescent quantitative PCR (Polymerase Chain Reaction) detection method for Lyme disease spirochetes
  • Nested fluorescent quantitative PCR (Polymerase Chain Reaction) detection method for Lyme disease spirochetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Design and optimization of nested fluorescent quantitative PCR primers

[0037] Borrelia Lyme disease has a unique rRNA gene structure, that is, there are two copies of 23S rRNA (rrf) and 5S rRNA (rrl) genes, which are repeated in sequence: -rrf-rrl-rrf-rrl-, forming rrf-rrl in the middle Intergenic region, this structure is specific to Borrelia burgdorferi.

[0038] The rrf-rrl site of Borrelia Lyme disease, which can be identified by single point typing, was selected as the target gene. In the first round, the outer round primers of nested PCR were used to enrich the DNA of Lyme disease spirochetes in the samples; in the second round, specific fluorescent quantitative PCR was used for the specific detection of Lyme disease spirochetes.

[0039] First round: common PCR

[0040] The primers are as follows (SEQ ID NO:1-2):

[0041] Upstream primer F1: 5'-CGACCTTCTTCGCCTTAAAGC-3'

[0042] Downstream primer R1: 5'-TAAGCTGACTAATACTAATTACCC-3'

[0043] The sec...

Embodiment 2

[0050] Example 2 Establishment of a nested fluorescent quantitative PCR detection method for Lyme spirochete

[0051] The nested fluorescent quantitative PCR detection method of Lyme disease spirochete provided by the invention comprises the following steps:

[0052] 1. Extract the DNA in the sample;

[0053] 2. Using the DNA extracted in step 1 as a template, use the first round of PCR primers (SEQ ID NO: 1-2) to carry out PCR amplification reaction;

[0054] 3. Using the amplified product in step 2 as a template, use the second round of PCR primers and probes (SEQ ID NO: 3-5) to carry out PCR amplification reaction;

[0055] 4. Analyze PCR products.

[0056] In step 2, the PCR reaction system is: 10 μL of reaction mixture, 0.5 μL of 10 μM upstream and downstream primers, 4 μL of DNA template, and 5 μL of deionized water.

[0057] The PCR reaction program is: 94°C for 2min, 55°C for 1min, 72°C for 2min, 1 cycle; 94°C for 45s, 55°C for 45s, 72°C for 45s, 33 cycles; 94°C for...

Embodiment 3

[0060] Example 3 Specificity investigation of nested fluorescent quantitative PCR detection method

[0061] The strains used included 22 samples of Escherichia coli (1 strain), Rickettsia (5 strains), Bartonella (1 strain), Leptospira (10 strains) and Borrelia burgdorferi (5 strains). The nested fluorescent quantitative PCR method in Example 2 was used for detection. The test results showed that, except Borrelia burgdorferi, other bacteria were not detected, indicating that the method had better specificity ( figure 1 ).

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Abstract

The invention provides a nested fluorescent quantitative PCR (polymerase chain reaction) detection method for Lyme disease spirochetes. According to the invention, rrf-rrl is taken as a target gene, nested PCR primers and probes for detecting lyme disease spirochetes are designed, the nested PCR primers comprise a first round of PCR primers and a second round of PCR primers, and the probes are introduced in the second round of PCR reaction so as to improve the detection sensitivity. The nested fluorescent quantitative PCR detection method for Lyme disease spirochetes provided by the invention has better specificity and high sensitivity, and the lower detection limit reaches 0.01 pg / [mu]L. According to the method disclosed by the invention, a positive sample can be subjected to single-point typing, so that the effective discrimination of four types of Borrelia burgdorferi, namely, Borrelia burgdorferi sensu stricto (B.b.s.s), Borrelia garinii (B.g), Borrelia afzelii (B.a), and Borrelia valaisiana (B.v) can be realized.

Description

technical field [0001] The invention relates to the technical field of molecular biology and microorganism detection, in particular to a nested fluorescent quantitative PCR detection method for 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 seriou...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6848C12Q1/04C12N15/11
CPCC12Q1/689C12Q1/6848C12Q2549/119C12Q2563/107Y02A50/30
Inventor 郝琴万康林季绍有范雪婷侯学霞张琳
Owner ICDC CHINA CDC
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