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LAMP primer combination for detecting 5 gram-positive bacteria in intraocular fluid and application thereof

A primer combination and primer set technology, applied in the biological field, can solve the problems of easy contamination, long PCR detection time and high false positive rate

Active 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 5 gram-positive bacteria in intraocular fluid and application thereof
  • LAMP primer combination for detecting 5 gram-positive bacteria in intraocular fluid and application thereof
  • LAMP primer combination for detecting 5 gram-positive bacteria in intraocular fluid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Embodiment 1, the preparation of kit

[0157] The kit consists of five LAMP primer sets, each for the detection of 1 Gram-positive bacterium in intraocular fluid.

[0158] The primer set used to detect Staphylococcus epidermidis is as follows (5'→3'):

[0159] Outer primer F3 (SEQ ID NO: 1): ATTgAgATAgCgggggA;

[0160] Outer primer B3 (SEQ ID NO: 2): ACAACAAAgTAACAgTACCATg;

[0161] Internal primer FIP (SEQ ID NO: 3): gCgTCATgCCTTTTATTTgAAgAAAATgTACAgTCATAgCTAgTggA;

[0162] Internal primer BIP (SEQ ID NO: 4): ACAggAgTAAATTCAgTgATTgCAATTCCgCAACTTACAAAACATg;

[0163] Loop primer LF (SEQ ID NO: 5): TTATATgTATgTgCCCAAAATCACA;

[0164] Loop primer LB (SEQ ID NO: 6): CCAATTgATTggAAAggATTTgATC.

[0165] The primer set used to detect Staphylococcus aureus is as follows (5'→3'):

[0166] Outer primer F3 (SEQ ID NO: 7): gCAACTgAAACAACAgAAgC;

[0167] Outer primer B3 (SEQ ID NO: 8): TTTTgTgTTgggCgAgC;

[0168] Internal primer FIP (SEQ ID NO: 9): TCACggATACCTgTACCAgCATCCTC...

Embodiment 2

[0193] Embodiment 2, specificity

[0194] Test sample 1: Staphylococcus epidermidis.

[0195] Test sample 2: Staphylococcus aureus.

[0196] Test sample 3: Enterococcus faecalis.

[0197] Test sample 4: Enterococcus faecium.

[0198] Test sample 5: Streptococcus pyogenes.

[0199] Each sample to be tested carries out the following steps respectively:

[0200] 1. Extract the genomic DNA of the sample to be tested.

[0201] 2. Using the genomic DNA extracted in step 1 as a template, each primer set prepared in Example 1 was used to perform loop-mediated isothermal amplification.

[0202] Reaction system (10 μL): 1 μL 10×ThermoPol Buffer, 1.6 μL betaine with a concentration of 5M, 0.1 μL BSA aqueous solution with a concentration of 50 mg / mL, and 0.4 μL MgSO with a concentration of 100 mM 4 Aqueous solution, 0.3 μL 20×EvaGreen, 0.15 μL of 100 mM dNTPs (each), 0.4 μL of 8 U / mL Bst DNA polymerase large fragment, genomic DNA of the sample to be tested (between 50 pg and 50 ng),...

Embodiment 3

[0212] Embodiment 3, sensitivity

[0213] Test sample 1: Staphylococcus epidermidis.

[0214] Test sample 2: Staphylococcus aureus.

[0215] Test sample 3: Enterococcus faecalis.

[0216] Test sample 4: Enterococcus faecium.

[0217] Test sample 5: Streptococcus pyogenes.

[0218] Each sample to be tested carries out the following steps respectively:

[0219] 1. Extract the genomic DNA of the sample to be tested, and perform gradient dilution with sterile water to obtain each dilution.

[0220] 2. Using the dilution obtained in step 1 as a template, the primer sets prepared in Example 1 were used to perform loop-mediated isothermal amplification.

[0221] When the sample to be tested is the sample to be tested 1, the loop-mediated isothermal amplification is performed using primer set I. When the sample to be tested is the sample to be tested 2, loop-mediated isothermal amplification is performed using primer set II. When the sample to be tested is the sample to be test...

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Abstract

The invention discloses an LAMP primer combination for detecting 5 gram-positive bacteria in intraocular fluid and an application thereof. The primer combination provided by the invention firstly is composed of 30 DNA molecules shown in a sequence 1 to 30. The primer combination can be applied for detecting whether to-be-tested bacteria are staphylococcus epidermidis, staphylococcus aureus, enterococcus faecalis, enterococcus faecium, or streptococcus pyogenes, and can be applied for detecting whether to-be-tested samples contain staphylococcus epidermidis and / or staphylococcus aureus and / or enterococcus faecalis and / or enterococcus faecium and / or streptococcus pyogenes. The primer combination provided by the invention is used for detecting 5 gram-positive bacteria in the intraocular fluid, has high specificity and sensitivity, can realize simple, rapid and accurate detection, and has great promotion value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a combination and application of LAMP primers for detecting five kinds of Gram-positive bacteria in intraocular fluid. Background technique [0002] Gram-positive bacteria are the main pathogens causing bacterial endophthalmitis. Gao Min et al. retrospectively analyzed 146 cases of keratitis patients with positive bacterial culture in Beijing Tongren Hospital from January 2010 to April 2015, among which 56.2% (82 cases) of pathogenic bacteria were Gram-positive bacteria. Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, Enterococcus Faecium and Streptococcus pyogenes are the five most common Gram species positive pathogenic bacteria. Staphylococcus epidermidis is a type of bacteria that grows on the epidermis of organisms. It belongs to the type of normal flora. Most of them are non-pathogenic bacteria. A few can cause diseases. Most bacterial en...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12Q1/14C12R1/45C12R1/445C12R1/46
CPCC12Q1/689C12Q1/6844C12Q2531/119Y02A50/30
Inventor 陶勇
Owner BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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