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Primer, method and kit for rapidly detecting lactococcus lactis

A technology of Lactococcus lactis and a detection method is applied in the field of primers for rapid and convenient detection of Lactococcus lactis, a detection method and a kit thereof, and achieves the effects of fast detection speed, high sensitivity and low cost

Inactive Publication Date: 2015-04-01
UNIV OF SHANGHAI FOR SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The problem is that the phenomenon of indiscriminate use in the market is relatively serious. Lactococcus lactis and Streptococcus lactis are not distinguished, and the phenomenon of mixed use and indiscriminate use is more prominent

Method used

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  • Primer, method and kit for rapidly detecting lactococcus lactis
  • Primer, method and kit for rapidly detecting lactococcus lactis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The detection of Lactococcus lactis in the yogurt of embodiment 1

[0027] Take 10ml of yogurt, centrifuge at 8000g for 10min, discard the supernatant, and take the precipitate. The precipitate was washed twice with deionized water, and the plasmid DNA of the bacteria was extracted with a plasmid extraction kit (SanPrep Column Plasmid DNA Extraction Kit, purchased from Sangon Bioengineering Co., Ltd.). Then PCR reaction was carried out using the extracted DNA as a template.

[0028] The PCR reaction system is: 0.5mmol / L upstream primer, 0.5mmol / L downstream primer, 0.2mmol / L dNTP, 1XPCR buffer, 1.5mmol / L Mg2+, 0.02U / μL Taq DNA polymerase, 1μL DNA template, add deionized water to 20μl. The PCR reaction program was: 95°C, 3min; 30 cycles (95°C 45s, 55°C 45s, 72°C 1min); 72°C 5min; 4°C storage.

[0029] Finally, electrophoresis detection was carried out, and 5 μl of the PCR product was electrophoresed in 1% agarose gel, the electrophoresis voltage was 100 V, and the ele...

Embodiment 2

[0031] Example 2 specificity verification

[0032] Select 20 different lactic acid bacteria strains, including 4 strains of Lactococcus lactis, 5 strains of Streptococcus lactis, 2 strains of Lactococcus plantarum, 2 strains of Lactococcus gasseri, 4 strains of Lactobacillus casei, and 3 strains of Lactobacillus plantarum. 50 other bacterial strains, including 10 strains of Escherichia coli, 10 strains of Bacillus subtilis, 10 strains of Staphylococcus aureus, 5 strains of Campylobacter, 5 strains of Salmonella, 5 strains of Pseudomonas, and 5 strains of Bacillus pumilus. Use the primer pair provided by the present invention to carry out PCR reaction, the specific operation of PCR amplification system and program and electrophoresis refer to Example 1.

[0033] The results showed that only Lactococcus lactis had a brighter 480bp electrophoresis band under ultraviolet light, and other strains and species had no 480bp electrophoresis band. It shows that the designed PCR primers...

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Abstract

The invention aims to provide a specific labeled molecule of lactococcus lactis. The specific labeled molecule is characterized by being a gene for coding the protein Usp45 of lactococcus lactis. The invention also aims to provide a primer pair for detecting lactococcus lactis. The primer pair is characterized in that one primer in the primer pair is the same as the specific labeled molecule of lactococcus lactis in sequence and has a length of 15-40bp, and the other primer in the primer pair is complementary to the specific labeled molecule of lactococcus lactis in sequence and has a length of 15-40bp, and the amplified fragment of the primer pair has a length of 200-1383bp. The specific labeled molecule of lactococcus lactis has the advantages of high detection speed, high specificity, high sensitivity and low cost.

Description

technical field [0001] The invention belongs to the field of biological detection, in particular to a primer for quickly and conveniently detecting Lactococcus lactis, a detection method and a kit thereof. Background technique [0002] Lactococcus lactis is the most representative strain in the genus Lactococcus. Lactococcus lactis has been identified as GRAS (Generally Recognized As Safe) by the US Food and Drug Administration and the World Health Organization, and is a fairly safe food microorganism. Therefore, it is often used in the food industry. Lactococcus lactis was originally identified as Streptococcus lactis and was not reclassified as Lactococcus lactis until 1987. At present, Lactococcus lactis includes 4 subspecies, namely Lactococcus lactis subsp. L. lactis subsp. hordniae) and L. lactis subsp. tructae. It has been found that all analyzed Lactococcus lactis strains contain a 45kDa secreted protein of unknown function, which is named Usp45. The discovery o...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 艾连中王光强夏永军陈卫王彦波郭本恒宋馨
Owner UNIV OF SHANGHAI FOR SCI & TECH
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