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LAMP primer combination for detecting candida albicans in intraocular fluid and application thereof

A Candida albicans, primer combination technology, applied in microorganism-based methods, microorganisms, recombinant DNA technology, etc., can solve the problems of easy contamination, high false positive rate, application limitations, etc., and achieve high specificity.

Pending Publication Date: 2019-08-16
BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, PCR has the disadvantages of long detection time, easy contamination, and high false positive rate, which limits its application.

Method used

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  • LAMP primer combination for detecting candida albicans in intraocular fluid and application thereof
  • LAMP primer combination for detecting candida albicans in intraocular fluid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Embodiment 1, primer design and preparation

[0058] A large number of sequence analyzes and comparisons were carried out to obtain several primers for identifying Candida albicans. Preliminary experiments were performed on each primer to compare performances such as sensitivity and specificity, and finally a LAMP primer set for identifying Candida albicans was obtained.

[0059] The primer set used to identify Candida albicans, including 2 outer primers (F3, B3), 2 inner primers (FIP, BIP) and 2 loop primers (LF, LB), each primer sequence is as follows (5 '→3'):

[0060] F3 (sequence 1 of the sequence listing): CgATACgTAATATgAATTgCAgAT;

[0061] B3 (sequence 2 of the sequence listing): CCATTgTCAAAgCgATCC;

[0062] FIP (SEQ ID NO: 3 of the Sequence Listing): AggCATgCCCTCCggAATACTCgTgAATCATCgAATCTTTgAAC;

[0063] BIP (SEQ ID NO: 4 of the Sequence Listing): gAgCgTCgTTTCTCCCTCAAACCgCCTTACCACTACCgTCTT;

[0064] LF (Sequence 5 of the Sequence Listing): AgAgggCgCAATgTgC;...

Embodiment 2

[0066] Embodiment 2, detection method establishment

[0067] 1. Extract the genomic DNA of the fungus to be tested or extract the total DNA of the biological sample to be tested.

[0068] 2. Take the DNA obtained in step 1 as a template, and use the LAMP primer set prepared in Example 1 to perform LAMP amplification.

[0069] Reaction system for LAMP amplification: 1μL 10×ThermoPol Buffer, 1.6μL 5M betaine, 0.1μL 50mg / ml BSA, 0.4μL 100mM MgSO 4 , 0.3 μL 20×EvaGreen, 0.15 μL 100 mM dNTPs, 0.4 μL 8U / ml Bst DNA polymerase large fragment, 1 μL primer mix (mixture of F3, B3, FIP, BIP, LF, and LB), 2ng template DNA, with ddH 2 O to make up to 10 μL. The concentration of each primer in the reaction system is as follows: 0.5 μM F3, 0.5 μM B3, 2 μM FIP, 2 μM BIP, 1 μM LF, 1 μM LB.

[0070] Reaction program for LAMP amplification: constant temperature at 65°C for 50min. During the reaction process, the fluorescent signal was detected by a fluorescent PCR instrument.

[0071] If a p...

Embodiment 3

[0072] Embodiment 3, specificity

[0073] Bacteria to be tested: Candida albicans, Aspergillus fumigatus, Aspergillus flavus and Candida glabrata.

[0074] The genomic DNA of the bacteria to be tested was extracted, and the detection method in Example 2 was used for detection. Set up a negative control using water as template DNA.

[0075] see results figure 1 . figure 1 In , the abscissa is the cycle number, and the ordinate is the fluorescence signal intensity. The results showed that Candida albicans genomic DNA obtained S-type amplification curve, and the amplification result was positive, while Aspergillus fumigatus genomic DNA, Aspergillus flavus genomic DNA and Candida glabrata genomic DNA did not obtain S-type amplification curve, and the amplification result was positive. is negative.

[0076] The results show that the LAMP primer set prepared in Example 1 has high specificity.

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Abstract

The invention discloses an LAMP primer combination for detecting candida albicans in intraocular fluid and an application thereof. The primer combination provided by the invention consists of six single-stranded DNA molecules shown in a sequence 1 to 6. The invention also provides the application of the primer combination in detection of candida albicans. The invention also provides a method for detecting whether a to-be-tested sample is infected with candida albicans. The LAMP primer and method can be applied for detecting candida albicans quickly and accurately.

Description

technical field [0001] The invention relates to a LAMP primer combination and application for detecting Candida albicans in intraocular fluid. Background technique [0002] Fungal endophthalmitis is an infectious eye disease that poses a great threat to vision. It has a high rate of blindness and a long incubation period, usually weeks or even months. The early clinical symptoms are not obvious, and the diagnosis is difficult. Therefore, clinically, especially in the early stage of the disease, it is often misdiagnosed and delayed treatment. The poor prognosis of fungal endophthalmitis is due to the insufficient efficacy of clinical antifungal drugs and large adverse reactions, and the strong destructiveness of fungi to ocular tissues. In recent years, with the massive abuse of corticosteroid hormones, antibiotics and immune preparations after surgery and chronic inflammation, the human body flora imbalance, immune function decline, and the incidence of fungal endophthalmit...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/725
CPCC12Q1/6895C12Q1/6844
Inventor 陶勇
Owner BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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