CN-122012762-A - Primer pair, kit and method for specifically detecting haemolyticus mannheimia

Abstract

The invention relates to the technical field of biology, and particularly discloses a primer pair, a kit and a method for specifically detecting haemolyticus managerial bacillus. The primer pair consists of an upstream primer shown in SEQ ID NO. 3 and a downstream primer shown in SEQ ID NO. 4. The primer provided by the invention has high specificity and sensitivity, and has the advantages of rapidness, simplicity and convenience in operation and the like, and the detection can be completed by carrying out 10 min-30 min under the constant temperature condition of 25-41 ℃, the detection sensitivity reaches 2.87 multiplied by 101 copies/mu L, the coincidence rate of the clinical sample detection result and qPCR reaches 98%, and the kit is suitable for rapid and accurate detection of the haemolytic mannich bacteria in basic laboratories and sites.

Inventors

• CUI YAOCHENG
• WANG SHU
• Xuan Huiyong
• WANG FANG
• QI YAYIN
• Cao Shuzhu

Assignees

• 石河子大学

Dates

Publication Date

20260512

Application Date

20260306

Claims (8)

1. 1. A primer pair for specifically detecting mannheimia haemolytica (MANNHEIMIA HAEMOLYTICA), characterized in that the primer pair comprises an upstream primer shown in SEQ ID No. 3 and a downstream primer shown in SEQ ID No. 4.
2. 2. A kit for specifically detecting mannheimia haemolytica, comprising the primer pair of claim 1.
3. 3. The kit of claim 2, further comprising one or more of a buffer, a reaction dry powder.
4. 4. A method for specifically detecting mannheimia haemolytica, comprising the steps of: Using the primer pair of claim 1 as template to perform amplification reaction in the recombinase-mediated isothermal nucleic acid amplification reaction system, wherein if a specific amplification band of 206 bp is generated, the sample to be tested contains the haemolyticus mannich bacillus.
5. 5. The method for specifically detecting the haemolyticus according to claim 4, wherein the recombinase-mediated isothermal nucleic acid amplification reaction system comprises 24-26 mu L of A Buffer, 12.5-14.5 mu L of ddH 2 O, 1.5-2.5 mu L of upstream primer, 1.5-2.5 mu L of downstream primer and reaction dry powder, and 4-6 mu L of DNA sample to be detected and 2-3 mu L of B Buffer are added when the reaction is started.
6. 6. The method for specifically detecting a mannheimia according to claim 5, wherein the concentration of the upstream primer and the concentration of the downstream primer are each 1. Mu. Mol/L to 10. Mu. Mol/L.
7. 7. The method for specifically detecting a mannheimia bacterium according to claim 4, wherein the detection amplification product is a specific band of 206 bp detected by agarose gel electrophoresis.
8. 8. The method for specifically detecting a mannheimia haemolytica according to claim 4, wherein the amplification reaction is carried out at a constant temperature of 25-41 ℃ for 10-30 min.

Description

Primer pair, kit and method for specifically detecting haemolyticus mannheimia Technical Field The invention relates to the technical field of biology, in particular to a primer pair, a kit and a method for specifically detecting haemolyticus mannhei. Background The haemolytic mannobacter (MANNHEIMIA HAEMOLYTICA, mh) is a gram-negative cocci, widely existing in the respiratory tract of ruminants such as cattle and sheep, and is a conditional pathogenic bacterium. When the immune function of animals is reduced due to long-distance transportation, feeding condition change, environmental stress or virus infection, mh can proliferate rapidly and descend to the lung, so that serious pneumonia is caused, and huge economic loss is caused for the breeding industry; meanwhile, as potential pathogenic bacteria of zoonosis, the haemolytic mannheimia has potential occupational exposure risk to people with low immune function or occupational contactors, and can easily pollute meat products in slaughter processing links, thereby causing public health safety and animal-derived food safety problems. Therefore, effective measures are needed to control the propagation of Mh, and rapid and accurate detection is a key to control Mh. The current commonly used Mh detection products comprise ELISA kits, PCR kits, real-time fluorescence quantitative PCR (qPCR) kits and the like, but the products have the problems of complex operation or low sensitivity, and are not suitable for laboratories or field detection with limited resources. Therefore, the development of the quick, simple and convenient Mh detection product with high sensitivity has important significance for effectively preventing and controlling Mh infection and reducing economic loss of the breeding industry. Disclosure of Invention The invention provides a primer pair, a kit and a method for specifically detecting haemolyticus mannhei. The primer provided by the invention has high specificity and sensitivity, and has the advantages of rapidness, simplicity and convenience in operation and the like, and the detection can be completed by carrying out 10 min-30 min under the constant temperature condition of 25-41 ℃, the detection sensitivity reaches 2.87 multiplied by 101 copies/mu L, the coincidence rate of the clinical sample detection result and qPCR reaches 98%, and the kit is suitable for rapid and accurate detection of the haemolytic mannich bacteria in basic laboratories and sites. The invention provides a primer pair for specifically detecting the haemolyticus mannhei (MANNHEIMIA HAEMOLYTICA), which comprises an upstream primer shown in SEQ ID NO.3 and a downstream primer shown in SEQ ID NO. 4. The primer provided by the invention has high specificity and sensitivity, and has no cross reaction with non-target pathogenic bacteria only when amplifying the specific strip to the haemolytic mannich bacillus. The invention also provides a kit for specifically detecting the haemolyticus mannhei, which comprises the primer pair. Further, the kit also comprises one or more of buffer solution and reaction dry powder. The invention also provides a method for specifically detecting the haemolyticus mannhei, which comprises the following steps: And (3) taking the DNA of the sample to be detected as a template, using the primer pair to perform amplification reaction in a recombinase-mediated isothermal nucleic acid amplification reaction system, and if a specific amplification band of 206 bp is generated, the sample to be detected contains the haemolyticus mannich bacillus. Further, the recombinase-mediated isothermal nucleic acid amplification reaction system comprises 24-26 mu L of A Buffer, 12.5-14.5 mu L of ddH 2 O, 1.5-2.5 mu L of upstream primer, 1.5-2.5 mu L of downstream primer and reaction dry powder, and 4-6 mu L of DNA sample to be detected and 2-3 mu L of B Buffer are added when the reaction is started. Further, the concentration of the upstream primer and the downstream primer is 1 mu mol/L to 10 mu mol/L. Further, the detection of the amplified product is to detect the presence or absence of a specific band of 206 bp by agarose gel electrophoresis. Further, the amplification reaction is carried out for 10 min-30 min under the constant temperature condition of 25-41 ℃. Compared with the prior art, the invention has the beneficial effects that: In a recombinase-mediated isothermal nucleic acid amplification (RAA) system, the primer pair provided by the invention has the advantages that the optimal reaction condition is that the nucleic acid amplification can be completed after the reaction is carried out for 20 minutes at the constant temperature of 37 ℃, and compared with the traditional PCR method which requires a thermal cycling process for several hours and the qPCR method which depends on a precise instrument, the detection time is obviously shortened, and the primer pair is more suitable for rapid detection. The RAA reaction is carried out in the constant-tem