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Primer group, detection method and kit for rapidly detecting Staphylococcus aureus by using LAMP technology and application

A detection kit and staphylococcus technology, applied in the field of microbial detection, can solve the problems of time-consuming, laborious, incompetent, and high detection cost, and achieve the effects of low equipment dependence, strong practicability, and good specificity

Pending Publication Date: 2021-10-22
武汉海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The bacterial culture method is the gold standard for the detection of pathogenic bacteria, but it takes 4-7 days to get the results. The operation is cumbersome, time-consuming and laborious, and has limitations in sensitivity and specificity. It can only detect live bacteria and requires skilled operating experience; Linked immunosorbent assay (ELISA) is better in specificity, but it needs to rely on professional equipment such as microplate reader
Various PCR techniques mainly analyze pathogenic bacteria through DNA extraction, PCR amplification, electrophoresis observation, or fluorescence curves. The detection is sensitive, accurate, and rapid, but it still relies on expensive PCR instruments, which has high detection costs and high requirements for experimental techniques. Competent on-site portable rapid detection

Method used

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  • Primer group, detection method and kit for rapidly detecting Staphylococcus aureus by using LAMP technology and application
  • Primer group, detection method and kit for rapidly detecting Staphylococcus aureus by using LAMP technology and application
  • Primer group, detection method and kit for rapidly detecting Staphylococcus aureus by using LAMP technology and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Establishment of Staphylococcus aureus-specific LAMP detection method

[0036] 1. DNA extraction from purely cultured bacteria

[0037] All the above-mentioned bacteria were inoculated in LB liquid medium overnight at 37°C under aerobic conditions. Take 1 mL of the bacterial liquid and use a commercial bacterial genome extraction kit to extract genomic DNA strictly according to the steps, and store the samples at -20°C.

[0038] 2. Fecal mock sample preparation and DNA extraction

[0039] Mix 300 μL of freshly cultured bacteria with 2 g of sterile normal mouse feces with PBS buffer, inoculate 100 μL of the suspension in LB liquid medium at 37 °C overnight, take 1 mL of the culture solution to extract genomic DNA according to the method in 1, and store the samples in at -20°C.

[0040] 3. LAMP reaction

[0041] The composition of 25 μL of the standard LAMP reaction system is as follows: 12.5 μL of 2×LAMP buffer, 1 μL of inner primer spA-FIP, 1 μL of inner ...

Embodiment 2

[0053] Embodiment 2 specific primer screening

[0054] In the present invention, a total of 19 LAMP primer sets were designed for the three target genes of nuC, spA, and mecA. Other primer sequences are shown in Table 2, wherein there are 12 spA gene primer sets, and each set of primers has outer primers F3 and B3 respectively. , internal primers BIP and FIP, with the same concentration of Staphylococcus aureus ATCC12600 genome as a template, using the same reaction conditions and amplification templates as in Example 1, carry out independent amplification detection respectively, the results are as follows figure 2 shown.

[0055] figure 2 In , different sequence numbers represent the primer set numbers of the corresponding genes. After 30 minutes of amplification, none of the 4 sets of LAMP primers for nuc had a positive result; only spA9 showed a clear blue color when amplified by the LAMP primers corresponding to the spA gene, which has a significant advantage compared ...

Embodiment 3

[0060] Embodiment 3 kit sensitivity test

[0061] Using the genome of Staphylococcus aureus ATCC12600 as a template, PCR amplification was carried out with primers spA-F3 and spA-B3, and the obtained product was recovered by commercial methods, and then serially diluted with enzyme-free water, from high to low, 1ng / μL, 10 -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL, 10 -6 ng / μL. Use this as a template for sensitivity testing, see real-time fluorescence amplification curves, color changes, and product electrophoresis results. image 3 .

[0062] image 3 A shows that there is a concentration-dependent relationship between the amplification time required for different concentrations of the target gene, and the concentration is 10 -6 It only takes about 40 minutes for the amplification curve to appear at ng / μL; image 3 B shows that the concentration range is 10 -6 ~1ng / μL of genomic nucleic acid, the kit of this application can produce a corresponding...

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Abstract

The invention belongs to the technical field of microbiological detection, and particularly relates to a primer group, a detection method and a kit for rapidly detecting Staphylococcus aureus by using an LAMP technology, and application. The Staphylococcus aureus virulence gene spA is selected as a specific target, four specific primer groups capable of amplifying the gene at 62-68 DEG C are designed, a detection result is indicated through a visual dye such that violet (negative) is changed into sky blue (positive). The method for detecting the Staphylococcus aureus is good in specificity, simple to operate, and free of expensive instruments, and the sensitivity is higher than that of conventional PCR. The kit disclosed by the invention can be used for rapidly and specifically detecting Staphylococcus aureus (within 1 hour), and the detection limit is 10<-6> ng / [mu]L. The kit is simple to operate, easy in result interpretation and suitable for import and export quarantine, food hygiene and clinical sample detection.

Description

technical field [0001] The invention belongs to the technical field of microbial detection, and in particular relates to a primer set, a detection method, a kit and an application for rapidly detecting Staphylococcus aureus by using LAMP technology. Background technique [0002] Staphylococcus aureus is a common foodborne pathogen. The metabolic type of Staphylococcus aureus is aerobic or facultative anaerobic, and has low requirements on the environment. 37°C is the optimum growth temperature, but it can still survive in various harsh environments. It can tolerate high salt and can tolerate up to 15 % concentration of NaCl solution, and has a certain tolerance to high temperature, and can survive for more than 30 minutes in a high temperature environment above 80 °C. Staphylococcus aureus can produce enterotoxin under appropriate conditions, causing food poisoning. In recent years, reports of food poisoning caused by Staphylococcus aureus have emerged in an endless stream...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/14C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2600/166C12Q2531/119C12Q2545/113C12Q2563/107C12Q2537/1376
Inventor 郑军平刘洪涛曹亚楠叶诚方海田张聪卞庆来
Owner 武汉海关技术中心
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