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LAMP detection kit for actinobacillus pleuropneumoniae

A detection kit and porcine pleuropneumonia technology, applied in the direction of microbial determination/inspection, recombinant DNA technology, microbial-based methods, etc., can solve the problems of limited practical application in pig farms, high technical requirements for testing personnel, expensive equipment, etc. problem, to achieve the effect of high accuracy, simple identification and high sensitivity

Pending Publication Date: 2020-08-25
XIAMEN YINXIANG GROUP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, various PCR-based detection methods have high sensitivity and good specificity, and are most widely used in laboratory detection; however, these methods require expensive instruments and equipment, high detection costs, and high labor costs for detection personnel. technical requirements, limiting their practical application in pig farms

Method used

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  • LAMP detection kit for actinobacillus pleuropneumoniae
  • LAMP detection kit for actinobacillus pleuropneumoniae
  • LAMP detection kit for actinobacillus pleuropneumoniae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Establishment of Actinobacillus pleuropneumoniae LAMP Detection Kit

[0035] LAMP-PCR detection kit for Actinobacillus pleuropneumoniae, including detection primer set, internal standard primer set, LAMP reaction solution, Bst DNA polymerase, positive control, negative control and internal standard.

[0036](1) detection primer set: take Actinobacillus pleuropneumoniae apxIVA gene as target gene, carry out the design of LAMP primer, described detection primer set comprises a detection outer primer pair, a detection inner primer pair and a detection loop primer pair, The detection outer primer pair consists of a forward detection outer primer shown in SEQ ID NO.01 and a reverse detection outer primer shown in SEQ ID NO.02, and the detection inner primer pair consists of the detection inner primer pair shown in SEQ ID NO.03. The forward detection inner primer shown in and the reverse detection inner primer shown in SEQ ID NO.04 are composed, and the detection lo...

Embodiment 2

[0054] Example 2 The LAMP detection method of Actinobacillus pleuropneumoniae

[0055] Utilize the LAMP detection kit of the Actinobacillus pleuropneumoniae of embodiment 1 to detect the sample, the steps are as follows:

[0056] (1) Extract the DNA of the sample to be tested.

[0057] (2) Utilize the LAMP primer composition of claim 1 to carry out LAMP constant temperature amplification to the sample DNA to be tested:

[0058] The 25μL reaction system for LAMP constant temperature amplification contains: apxIVA-F3 0.2μM, apxIVA-B3 0.2μM, apxIVA-FIP 1.2μM, apxIVA-BIP 1.2μM, apxIVA-LF 0.6μM, apxIVA-LB 0.6μM, omlA-F3 0.2 μM, omlA-B 30.2μM, omlA-FIP 1.1μM, omlA-BIP 1.1μM, omlA-LoopF ​​0.6μM, omlA-LoopB 0.6μM, LAMP reaction solution 12.5μL, DNA polymerase 8U, 10×SYBR Green I 0.5μL , 2 μL of the sample to be tested, 2 μL of the internal standard, make up to 25 μL with ultrapure water;

[0059] The program of LAMP constant temperature amplification is: react at 63-65°C for 30-60m...

Embodiment 3

[0065] Embodiment 3 Sensitivity experiment

[0066] The constructed plasmid was subjected to a sensitivity test, and the 1pg / μL plasmid was diluted 10 times into 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL, 0.1fg / μL and other gradients as Quality control standard, detect with the method of embodiment 2, determine the minimum detection limit 1fg / μL of internal standard by sensitivity experiment (such as figure 1 Shown), and review the minimum detection limit ( figure 2 ), the internal standard concentration was set as the concentration of the lowest detection limit.

[0067] The Actinobacillus pleuropneumoniae was cultured, the cultured bacteria were diluted 10 times, and the DNA was extracted. At the same time, the diluted bacterial liquid was counted on the plate culture, and the plate culture count results were compared with the lowest sensitivity of this kit. The minimum detection degree of this kit is 1.2×10 2 CFU / mL, test results see image 3 .

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Abstract

The invention discloses an LAMP detection kit for actinobacillus pleuropneumoniae. The LAMP detection kit is provided with a detection primer group and an internal standard primer group, wherein the detection primer group comprises a detection outer primer pair, a detection inner primer pair and a detection loop primer pair, and the internal standard primer group comprises an internal standard outer primer pair, an internal standard inner primer pair and an internal standard loop primer pair. The LAMP detection kit has the beneficial effects of rapidness, high efficiency, simplicity and convenience in operation, high specificity, high sensitivity, low cost, no need of expensive instruments, suitability for field detection and the like, and more importantly, a use method improves the detection accuracy, false negative results in detection can be discovered in time, and various accidents caused by the false negative detection result can be effectively prevented.

Description

technical field [0001] The invention belongs to the technical field of molecular diagnosis, and in particular relates to a LAMP detection kit for Actinobacillus pleuropneumoniae. Background technique [0002] Actinobacillus pleuropneumoniae (App) can cause porcine contagious pleuropneumonia, and its pathological features are fibrinous pleurisy and hemorrhagic and necrotizing encephalitis. The disease is internationally recognized as one of the five major diseases that endanger the modern pig industry, and has caused huge economic losses to the pig industry in various countries. The morbidity and mortality rate of this disease is extremely high, but in most cases, it is a recessive infection. According to reports, pigs with healthy clinical performance still have pathogen parasites in their upper respiratory tract. This is the main reason for the spread of Actinobacillus pleuropneumoniae, and it is also the main reason for the decreased weight gain rate, decreased feed conve...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12N15/11C12R1/01
CPCC12Q1/6844C12Q1/689C12Q2600/166C12Q2531/119C12Q2563/107
Inventor 张志刚石磊苏永裕
Owner XIAMEN YINXIANG GROUP
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