A kind of pullorum salmonella detection kit and using method thereof

A detection kit and technology for Salmonella, applied in biochemical equipment and methods, microbiological determination/inspection, DNA/RNA fragments, etc., can solve the problems of rapid detection and diagnostic methods with less reports

Inactive Publication Date: 2021-09-10
SICHUAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The single-plex and multiplex PCR methods established for Salmonella at home and abroad are mostly used to detect the serotype, virulence factors or drug resistance genes of the strains, but there are few reports on the rapid detection and diagnosis methods for Salmonella pullorum in the Salmonella genus

Method used

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  • A kind of pullorum salmonella detection kit and using method thereof
  • A kind of pullorum salmonella detection kit and using method thereof
  • A kind of pullorum salmonella detection kit and using method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Salmonella pullorum specific target primer primer design and testing

[0042] 1. Primer Design

[0043]Download the sequences in the NCBI GenBank database: pSPUV(JN885081), pSGAV(CM001154.1), pSDUV(NC_007208.1), pSCSV(NC_006855.1), pSPCV(NC_012124.1), p14028S(CP001362.1) and pSLT( NC_003277.1). Conserved and specific regions between pSPUV and other VPs were searched by MAUVE program. The degree of divergence was calculated by the SITES program with a window size of 1000 bp and a step size of 100 bp. Combined with manual adjustment, detection primers were designed in the specific region of Salmonella pullorum virulence plasmid pSPUV.

[0044] The primer pairs in Table 1 were obtained: traJ-387-F / R, traJ-476-F / R and ipaJ-417-F / R, hereinafter referred to as traJ-387, traJ-476 and ipaJ-417.

[0045] 2. Primer Validation

[0046] Take 200 μl of the bacterial liquid of the verified strain, centrifuge at 10,000r / min for 3min, discard the supernatant, wash with 1...

Embodiment 2

[0052] Embodiment 2 buffer debugging

[0053] 1. Method

[0054] Select two different types of DNA polymerase combinations, select DNA polymerase Tfl and KOD according to the commercially available enzyme user manual and presentation information, and based on the reference information of the reaction buffer components of the two enzymes, comprehensively consider the performance of each component in PCR. For the contribution level of specificity and reaction efficiency in the reaction, prepare the reaction buffer system according to Table 2 and carry out PCR respectively, and screen the shared reaction buffer for the two DNA polymerases Tfl and KOD according to the amplification effect.

[0055] Table 2 Different reaction buffers of DNA polymerase

[0056]

[0057] 2. Results

[0058] Two kinds of different DNA polymerase Tfl and KOD are carried out PCR amplification effect comparison ( figure 2 ), both buffer 3# and 7# can make DNA polymerases belonging to different fam...

Embodiment 3 3

[0060] Example 3 Triple PCR Primer Screening

[0061] The triple PCR primers consisted of PCR system verification primers (16S rDNA primers), Salmonella-specific primers, and Salmonella pullorum-specific primers verified in this paper (Table 1). Combination screening was performed according to different fragment lengths and amplification efficiencies.

[0062] 1. Sample pretreatment

[0063] Centrifuge the Salmonella pullorum liquid at 10,000r / min for 2min, discard the supernatant, resuspend the bacteria in 50μl sterile water, boil for 10min, and finally centrifuge at 12,000r / min for 2min, then use the supernatant as a PCR template.

[0064] 2. Reaction system

[0065] A 25 μl system was selected as the PCR system, and the components are shown in Table 3. Basic PCR reaction conditions: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 56°C annealing for 20 seconds, and 72°C extension for 1 minute and 20 seconds, a total of 30 cycles. According to the am...

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Abstract

The invention provides a Salmonella pullorum detection kit, which comprises: Salmonella pullorum specific primers, PCR buffer, PCR enhancer, dNTP, DNA polymerase Tfl and DNA polymerase KOD; the sequence of the Salmonella pullorum specific primers As shown in SEQ ID NO.11-12; the PCR buffer contains 200-260 molar parts of Tris-HCl, 4-8 molar parts of MgCl 2 , 50‑120 molar parts of (NH4) 2 SO 4 , 90-110 molar parts of KCl and 1-3 parts by volume of Triton X-100; when the amount of KCl was 90-110mmol, the amount of Triton X-100 was 1-3ml. The present invention also provides a corresponding use method, and the corresponding dosage ratio of polymerase Tfl and DNA polymerase KOD is (1-30):1. The invention can specifically and sensitively detect Salmonella pullorum and other Salmonella, and has a good application prospect in the prevention and control of poultry diseases.

Description

technical field [0001] The invention relates to the field of bacterial detection, in particular to a detection kit for Salmonella pullorum and a detection method for Salmonella pullorum. Background technique [0002] Salmonella is a common and important zoonotic pathogen. According to the difference in the invasiveness of Salmonella to different hosts, Salmonella can be divided into widespread Salmonella and host-specific Salmonella. Chicken is the main host of Salmonella. Salmonella pullorum and Salmonella gallinarum only infect chickens and turkeys. They have high host adaptability and host tropism, and can cause acute sepsis in chicks, resulting in high morbidity and mortality. Pullorum is caused by the infection of chickens with Salmonella pullorum serotype. It mainly exists in ovaries, testes, liver and other organs, and can be transmitted to chicks from infected parental chickens. The mortality rate of chicks under 2 weeks after onset can reach 40%-70%, infected chick...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/686C12Q1/10C12N15/11
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2565/125Y02A50/30
Inventor 白林含黄非
Owner SICHUAN UNIV
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