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Chicken poison mycoplasma fluorescent quantitative detection kit and detection method thereof

A fluorescent quantitative detection, Mycoplasma gallisepticum technology, applied in the directions of microorganism-based methods, microorganism determination/inspection, biochemical equipment and methods, etc. Infection and other problems, to achieve the effect of simple pretreatment process, high repeatability and good specificity

Inactive Publication Date: 2017-02-15
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Mycoplasma gallisepticum infection mainly manifests as chronic respiratory symptoms without other characteristic clinical symptoms, and it is easy to be secondary and mixed with other bacteria or viruses, which brings difficulties to clinical diagnosis
Mycoplasma culture has high nutritional requirements, in vitro culture grows slowly and takes a long time, so the traditional separation and culture method cannot meet the needs of clinical diagnosis. At present, molecular biology methods are more and more widely used in clinical diagnosis due to their fast and sensitive characteristics.

Method used

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  • Chicken poison mycoplasma fluorescent quantitative detection kit and detection method thereof
  • Chicken poison mycoplasma fluorescent quantitative detection kit and detection method thereof
  • Chicken poison mycoplasma fluorescent quantitative detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] (1) Design of primers

[0032] Primers were designed according to the published Mycoplasma gallisepticum (MG) 16S rRNA gene sequence (Genbank accession number KC995374.1) registered in Genbank. Through analytical experiments, the following primers were obtained:

[0033] Mycoplasma gallisepticum - upstream primer: 5'-GAGCTAATCTGTAAAGTTGGTC-3'

[0034] M. gallisepticum - upstream primer: 5'-GCTTCCTTGCGGTTAGCAAC-3'.

[0035] (2) Handling of samples to be tested

[0036] a. Take a sterilized 1.5mL centrifuge tube, add 400μL sterile PBS, spin and wash the collected throat swabs in PBS, and keep the washing liquid;

[0037] b. Centrifuge the above washing solution at 12000r / min for 10min, discard the supernatant, and keep the precipitate;

[0038] c. Add the precipitate to 400 μL DEPC water and mix well, boil in a water bath at 100°C for 10 minutes, then ice-bath for 10 minutes after boiling, centrifuge at 2000 r / min for 3 minutes, and use the supernatant as the sample t...

Embodiment 2

[0046] Embodiment 2 Mycoplasma gallisepticum fluorescence quantitative PCR detection

[0047] (1) The above-mentioned PCR amplification reaction system was subjected to fluorescent quantitative PCR detection, and the parameters of the fluorescent quantitative PCR reaction were designed as follows: the first stage: pre-denaturation, 95°C for 3min; the second stage: 95°C for 15s, 59°C for 40s, 40 Cycling, fluorescence was collected during the 59°C annealing extension of each cycle in the second stage; in the third stage, melting curves were generated.

[0048] (2) Read the fluorescence data of the sample detection channel, positive control detection channel and negative control detection channel respectively, and judge the results: the negative control detection channel should have no Ct value and no amplification curve, and the positive control detection channel should have characteristic A positive "S" type amplification curve, and the Ct value is less than 32, and the Tm valu...

Embodiment 3

[0052] Embodiment 3 feasibility experiment

[0053] With the inactivated Mycoplasma gallisepticum live vaccine as a sample, the method of Example 1 and Example 2 was used to detect Mycoplasma gallisepticum by fluorescent quantitative PCR, and the test results showed that the sample had a typical amplification curve, the Ct value was less than 32, and the Tm value It is 84.1°C. It shows that the kit and detection method of the present invention have definite feasibility.

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Abstract

The invention belongs to the technical field of animal quarantine, and particularly relates to a chicken poison mycoplasma fluorescent quantitative detection kit and a detection method thereof. The detection kit comprises a fluorescent quantitative PCR reaction liquid, a positive control and a negative control, wherein the fluorescent quantitative PCR reaction liquid comprises an upstream primer, a downstream primer and SYBR Premi*E*Taq (Tli RNaseH Plus). By adopting the detection kit to conduct chicken poison mycoplasma fluorescent quantitative PCR detection on samples, the preprocessing process of the samples is very simple, there is no need to conduct mycoplasma cultivation on the samples or conduct mycoplasma gene extraction on the samples, and cooling after direct boiling of the samples is only required, so that both time and cost are saved. The detection kit has the advantages of being high in detecting sensitivity, good in specificity and repeated stability, and is very applicable to the detection of the chicken poison mycoplasma in the chicken throat swabs.

Description

technical field [0001] The invention belongs to the technical field of animal quarantine, and in particular relates to a fluorescent quantitative detection kit for mycoplasma gallisepticum and a detection method thereof. Background technique [0002] Mycoplasma gallisepticum (Mycoplasma gallisepticum, MG) infection is a kind of chronic respiratory disease in chickens. The clinical symptoms of chickens infected with MG include coughing, sneezing, rhinitis, conjunctivitis, accompanied by nasal secretions when breathing, and may also suffer from unilateral or bilateral sinusitis. After infection, it will cause the laying rate of laying hens to decrease and the weight of broilers to lose weight. The infectivity and pathogenicity of different strains of Mycoplasma gallisepticum are different, and sometimes the infectivity is not obvious. The mode of transmission is mainly vertical transmission and horizontal transmission. In 1943, Delaplane et al. isolated the pathogen from inf...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12R1/35
CPCC12Q1/6851C12Q1/689C12Q2531/113C12Q2563/107
Inventor 徐高原杨应立李冉李淑云杨惠佳万小祥贾运强
Owner WUHAN KEQIAN BIOLOGY CO LTD
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