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Kit for rapid test of pseudomonas aeruginosa

A Pseudomonas aeruginosa and kit technology, applied in the field of molecular biology, can solve the problems of inability to meet the needs of rapid and simple instant detection, high requirements for experimental conditions, and high maintenance costs, and achieve real-time detection of Pseudomonas aeruginosa. , The amplification compatibility is strong, and the effect of eliminating pollution

Pending Publication Date: 2019-12-17
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The nucleic acid extraction process generally uses kits based on various principles such as guanidinium salt lysis method, silicon membrane method, and magnetic bead method, or an automatic nucleic acid extraction instrument. However, it is time-consuming and laborious to extract nucleic acids with kits, and the operation is complicated. It often takes 2 hours. The above time cannot meet the needs of fast and simple instant detection. The automatic nucleic acid extraction instrument can achieve high throughput and is easy to operate, but it is cumbersome and has high maintenance costs.
[0006] Therefore, based on the shortcomings of insufficient sensitivity and high requirements for experimental conditions in the existing methods for detecting Pseudomonas aeruginosa, it is urgently needed by those skilled in the art to provide a sensitive, simple and rapid detection kit for Pseudomonas aeruginosa. solved problem

Method used

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  • Kit for rapid test of pseudomonas aeruginosa
  • Kit for rapid test of pseudomonas aeruginosa
  • Kit for rapid test of pseudomonas aeruginosa

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Primer design

[0080] According to the Pseudomonas aeruginosa toxA gene sequence published by NCBI (Reference Sequence: NC_002516.2), based on the principle of enzyme strand displacement activity, a specific primer pair for rapid detection of Pseudomonas aeruginosa, and the nucleoside of the primer pair were designed The acid sequence is as follows:

[0081] Ft: 5'- gcatcgtcttcggcggggt GGAGCGCGGCTATGTGT-3'; SEQ ID NO.1;

[0082] Bt: 5'- gcatcgtcttcggcggggt TCGGGTTCCTGGTCCTG-3'; SEQ ID NO. 2.

[0083] In this embodiment, Chelex-100, betaine, Sigma company; Manganese chloride, magnesium sulfate, potassium chloride, sodium hydroxide, EDTA, ammonium sulfate, Sinopharm Chemical Reagent Co., Ltd.; Tris-HCl, Shanghai Shengke Biotechnology Co., Ltd.; TritonX-100, Beijing Meilaibo Medical Technology Co., Ltd.; dNTP, Pharmacia Company; NP-40, Fluka Company; 2×TapMIX kit, Tiangen Biochemical Technology (Beijing) Co., Ltd.; Agarose, Amresco Company.

[0084] The reagent in th...

Embodiment 2

[0090] Accelerate the introduction of primers

[0091] If only two primers are used, the peaking time is generally after 50 minutes, which cannot meet the requirements of rapid and sensitive on-site detection. The introduction of accelerated primers includes IF and IB, respectively, between F and N, B and N, starting from the 5' end The direction to the 3' end is opposite to Ft and Bt respectively, and the schematic diagram of the positions of IF and IB on the target sequence is shown in figure 2 .

[0092] In this example, the accelerating primer is IF: 5'-AAGGTGCCGTGGTAGCC-3'; SEQ ID NO.3;

[0093] IB: 5'-TCTATATCGCCGGCGATCC-3'; SEQ ID NO.4.

[0094] The reagents in the kit of the present invention are stored using a one-pack diagnostic reagent bottle, wherein the reagent bottle is provided with a detection tube 1, a detection tube cover 3, a placement groove 4 and a coating 2; wherein the detection tube 1 is open at one end , a hollow reaction tube with the other end cl...

Embodiment 3

[0098] Betaine concentration is verified on the basis of Example 2.

[0099] Set seven betaine concentrations (working concentrations) from 0.5M to 1.1M with a spacing of 0.1 to investigate the influence of different betaine concentrations on the reaction Ct value.

[0100] Detected by turbidity method, the results are as follows Figure 4 As shown, No. 3 curve (0.7M) and No. 4 curve (0.8M) have the fastest reaction peak rate, but the peak concentration of betaine at 0.8M is slightly higher than 0.7M, and finally 0.8M is used as its optimum concentration.

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Abstract

The invention discloses a kit for rapid test of pseudomonas aeruginosa. The kit comprises specific primer pairs and the kit containing the specific primer pairs, wherein the kit comprises a first reagent, a second reagent and a sealing agent; the first reagent comprises Ft, Bt, IF, IB, dNTP, BstDNA polymerase and an indicator; and the second reagent comprises (NH4)2SO4, KCl, MgSO4, Tween20 and glycine betaine, and pH is 8.0. According to the kit provided by the invention, a special buffering system is adopted, and has the advantages that amplification compatibility is high, impurities such asprotein do not need to be removed thoroughly, efficient specific amplification can be conducted, and the kit meets inspection detecting requirements of quarantine on the spot under background of largepeople flow and faster customs clearance, and is also suitable for rapid test in a laboratory.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for rapidly detecting Pseudomonas aeruginosa. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas Aeruginosa), commonly known as Pseudomonas aeruginosa, is ubiquitous in the environment, especially in humid environments. Pseudomonas aeruginosa is a common opportunistic pathogen and one of the main pathogens of nosocomial infection. It often causes postoperative wound infection, and can also cause bedsores, abscesses, and suppurative otitis media. Infection lesions caused by the bacteria can lead to hematogenous dissemination, followed by bacteremia and sepsis, therefore, infection with Pseudomonas aeruginosa after burns may cause death. Those who are susceptible to this bacterium are those suffering from metabolic diseases, hematological diseases and malignant tumors, as well as patients after surgery or certain treatments. The bacterium can be passed on to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/385
CPCC12Q1/689
Inventor 王静张晓龙刘威张乔慈颖施琦杨燕孙筱霞
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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