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A pair of primers for simultaneous rapid detection of Streptococcus agalactiae and Streptococcus iniae and its application

A technology for Streptococcus iniae, Streptococcus nisella, applied in microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of high cost, primer interference, complicated operation steps, etc., and achieves simple operation and high accuracy. , The effect of low detection cost

Active Publication Date: 2019-04-05
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these technologies also have certain deficiencies, such as 16S sequencing technology is difficult to distinguish species with high homology; fluorescent antibody technology and ELISA are expensive and have complicated operation steps; LAMP is prone to false positives; quantitative PCR requires expensive instruments , professional operation and high cost; there will be interference between two or more pairs of primers in double and multiplex PCR, etc.

Method used

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  • A pair of primers for simultaneous rapid detection of Streptococcus agalactiae and Streptococcus iniae and its application
  • A pair of primers for simultaneous rapid detection of Streptococcus agalactiae and Streptococcus iniae and its application
  • A pair of primers for simultaneous rapid detection of Streptococcus agalactiae and Streptococcus iniae and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Establishment of Rapid Detection of Streptococcus agalactiae and Streptococcus iniae simultaneously

[0039] (1) Template preparation: take purely cultured Streptococcus agalactiae, Streptococcus iniae, Streptococcus dysgalactiae, Staphylococcus aureus, Aeromonas hydrophila, Edwardsiella tarda, Vibrio vulnificus, Vibrio alginolyticus and Escherichia coli Bacillus, using a DNA extraction kit (Dalian Bao Biological Engineering Co., Ltd.) to extract its genomic DNA, used as a reaction template;

[0040] (2) Design and synthesis of primers: According to the literature, download the whole genome sequences of Streptococcus agalactiae and Streptococcus iniae from NCBI, and find two homologous sequences through comparison, and the size between the homologous sequences is within two Bacteria are different and of the right size. Finally, it was found that the 16S and 23S of Streptococcus agalactiae and Streptococcus iniae had high homology, and the size of the spacer s...

Embodiment 2

[0047] Embodiment 2 A kind of DNA sensitivity of rapid detection Streptococcus agalactiae and Streptococcus iniae method simultaneously, comprises the steps:

[0048] (1) Separately extract the DNA of Streptococcus agalactiae and Streptococcus iniae: Take 2-3 mL of purely cultured Streptococcus agalactiae and Streptococcus iniae respectively, use a commercial DNA extraction kit to extract DNA, and use a spectrophotometer measure its concentration;

[0049] (2) Using the system and procedures of Example 1, the DNA of Streptococcus agalactiae and Streptococcus iniae were used as templates for positive controls, and DEPC water was used as templates for negative controls;

[0050] (3) Dilute the DNA concentration of the measured concentration to 1.5μg / μL, 1.5μg / μL, 150ng / μL, 15ng / μL, 1.5ng / μL, 0.15ng as required, Streptococcus agalactiae and Streptococcus iniae / μL, 15pg / μL, 1.5pg / μL, 0.15pg / μL and 15fg / μL;

[0051] (4) if figure 2 a and image 3 As shown in a, the DNA sensit...

Embodiment 3

[0052] Embodiment 3 A kind of single bacterium sensitivity of rapid detection Streptococcus agalactiae and Streptococcus iniae method simultaneously, comprises the steps:

[0053] (1) Pick Streptococcus agalactiae and Streptococcus iniae on the plate respectively, wash twice with PBS (phosphate-buffered saline, pH7.4), dilute in a ten-fold gradient, and count on the plate;

[0054] (2) Streptococcus agalactiae and Streptococcus iniae were diluted to 5.8×10 5 , 5.8×10 4 , 5.8×10 3 , 5.8×10 2 , 5.8×10 1 , 5.8×100, 5.8×10 -1 , 5.8×10 -2 , 5.8×10 -3 and 5.8×10 -4 CFU / mL;

[0055] (3) Using the system and procedures of Example 1, the DNA of Streptococcus agalactiae and Streptococcus iniae were used as templates for positive controls, and DEPC water was used as templates for negative controls; The DNA extraction kit of the DNA extraction kit extracts the DNA of the sample to be tested as a template; you can also directly boil the bacterial liquid for 10 minutes, and take th...

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Abstract

The invention discloses a primer capable of simultaneously and quickly detecting streptococcus agalactiae and streptococcus iniae and application of the primer. The primer disclosed by the invention is used for detecting a sample to simultaneously and specifically detect the streptococcus agalactiae and the streptococcus iniae in the sample at one time. Lower detection limits of a molecular detection method established according to the invention are both 1.5 pg / [mu]L and 5.8 CFU / [mu]L; the primer has the sensitivity of 105 CFU / g to both bacteria in mixed tilapia mossambica tissue samples respectively artificially infected with the streptococcus agalactiae and the streptococcus iniae; the primer can be detected in tissue organs such as the brain, the liver, the spleen and the kidney of tilapia mossambica injected with the streptococcus agalactiae and the streptococcus iniae through the abdominal cavity. The method can simultaneously and quickly detect the streptococcus agalactiae and the streptococcus iniae during tilapia mossambica aquaculture, does little harm to a detected object, is quick and accurate, is easy and convenient to operate, and can realize early molecular prewarningof tilapia streptococcus diseases.

Description

technical field [0001] The invention relates to the detection technology of target DNA fragments, in particular to a pair of primers for simultaneous rapid detection of Streptococcus agalactiae and Streptococcus iniae and application thereof. Background technique [0002] Tilapia is one of the most widely farmed fish species in the world, with global production reaching 4.5 million tonnes in 2013 alone. Although tilapia tolerates poor water quality better than other freshwater fish, with extensive aquaculture operations, the aquaculture industry is affected by a variety of pathogens, most notably Streptococcus. The pathogenic bacteria of streptococcosis are mainly Streptococcus agalactiae and Streptococcus iniae, which have the characteristics of high morbidity and mortality, and have caused huge economic losses. [0003] Streptococcus agalactiae and Streptococcus iniae are difficult to distinguish and identify, especially because they have high homology, so it is easy to c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/14C12N15/11C12R1/46
CPCC12Q1/689
Inventor 崔淼张辉杰张其中许德麟
Owner JINAN UNIVERSITY