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Dual fluorescent PCR primer group, kit and method for detecting salmonella enteritidis and mycoplasma synoviae

A technology for Mycoplasma gallisepticum and Salmonella synovialis, which is applied in the field of animal pathogen detection, can solve the problems of inability to detect Salmonella gallisepticum and Mycoplasma synovialis dual real-time fluorescent PCR, single detection of Salmonella gallisepticum or Mycoplasma synovialis, time-consuming and labor-intensive, etc. , to achieve the effect of improving operation convenience, good specificity, and convenient promotion and application.

Pending Publication Date: 2022-06-03
SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these conventional methods are cumbersome to operate, time-consuming, labor-intensive, low-sensitivity, poor specificity, and there is a certain probability of missed detection and false positives. More sensitive and specific high-efficiency screening kits for two pathogens are urgently needed to be developed
At present, among the two pathogen detection methods, the established real-time fluorescent PCR detection method mainly for Salmonella gallinarum, or the real-time fluorescent PCR detection method for Mycoplasma synovialum, can only detect Salmonella gallinarum or Mycoplasma synovialum, but cannot Duplex real-time fluorescent PCR detection of Salmonella enteritidis and Mycoplasma synoviae

Method used

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  • Dual fluorescent PCR primer group, kit and method for detecting salmonella enteritidis and mycoplasma synoviae
  • Dual fluorescent PCR primer group, kit and method for detecting salmonella enteritidis and mycoplasma synoviae
  • Dual fluorescent PCR primer group, kit and method for detecting salmonella enteritidis and mycoplasma synoviae

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Establishment of dual fluorescent PCR reaction system for Salmonella enteritidis and Mycoplasma synovium

[0044] 1. Primer design and screening

[0045] Find the sefA gene sequence of different strains of Salmonella enteritidis and the vlhA gene sequence of different strains of Mycoplasma synovialis from GenBank, select the conserved regions and design 4 groups of primers after comparison and analysis, and the 4 groups of primers are group A, group B, group C respectively And group D, all primers are about 20 bases in length, GC content is 40% to 60%, there is no complementary sequence between primers, the difference in melting temperature is less than 5 °C, and there is no complementary sequence and secondary structure in the primers. Synthesized by System (Anhui) Co., Ltd. The sequences of the 4 sets of primers are as follows:

[0046] The specific nucleotides of group A primers are as follows:

[0047] SE-F1: GGGCTTCGGTATCTGGTG (SEQ ID NO. 1);

[0048] SE-R1: GG...

Embodiment 2

[0084] The composition of the double fluorescent PCR detection kit for Salmonella enteritidis and Mycoplasma synovialis

[0085] 1. PCR reaction solution A: each reaction includes reaction buffer Green Pro Taq HS Premix (with MgCl 2 , dNTP, SYBR Green I and Taq enzyme) 13.8 μL, aliquoted into 1 mL serum tubes, 800 μL / tube, and stored at -20°C;

[0086] 2. PCR reaction solution B: each reaction consists of 1.2 μL SE-F1 (10 μmol / L), 1.5 μL SE-R1 (10 μmol / L), 0.8 μL MS-F1 (10 μmol / L), 0.7 μL MS-R1 (10μmol / L), a total of 4.2μL, aliquoted into 0.5mL serum tubes, 220μL / tube, and stored at -20°C;

[0087] 3. Positive control: The positive control plasmids pMD-sefA and pMD-vlhA were mixed in a volume ratio of 1:1, 300 μL / tube, and stored at -20°C.

[0088] 4. Negative control: sterile deionized water, 300 μL / tube, stored at -20°C.

[0089] 5. Nucleic acid release agent: 10ml / bottle, dispense into white and transparent plastic bottles with white caps, and store at room temperature...

Embodiment 3

[0093] How to use the double fluorescent PCR detection kit for Salmonella enteritidis and Mycoplasma synovialis

[0094] Throat cotton swabs and cloacal swabs were collected from each chicken and placed in the same centrifuge tube containing PBS as one sample, vortexed, and 100 μL was added with an equal volume of nucleic acid release agent, placed at room temperature for 5 min, and centrifuged at 12000 r / min for 1 min. Taking the supernatant as a template, the kit of the present invention is used for double fluorescent PCR detection of Salmonella enteritidis and Mycoplasma synovialis. 20 μL of reaction system: 13.8 μL of PCR reaction solution A, 4.2 μL of PCR reaction solution B, and 2 μL of template. After a brief centrifugation, the amplification reaction was carried out in a fluorescence PCR instrument. The reaction program was: pre-denaturation at 95°C for 2 min, denaturation at 94°C for 5s, and annealing at 55°C for 20s (the fluorescence was collected here) for a total ...

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Abstract

The invention discloses a dual fluorescence PCR primer group, a kit and a method for detecting salmonella enteritidis and mycoplasma synoviae, the primer group provided by the invention comprises specific primers aiming at salmonella enteritidis and mycoplasma synoviae, the length of each primer is about 20 basic groups, the GC content is 40-60%, no complementary sequence exists among the primers, and the specific primers can be used for detecting the salmonella enteritidis and mycoplasma synoviae in the salmonella enteritidis and mycoplasma synoviae in the salmonella enteritidis and mycoplasma synoviae in the salmonella enteritidis and mycoplasma synoviae. The melting temperature difference is less than 5 DEG C, and no complementary sequence or secondary structure exists in the primer. The kit provided by the invention simultaneously contains the primer groups of the salmonella enteritidis and the mycoplasma synoviae, the four primers do not interfere with one another during amplification, two pathogens can be screened by one-time amplification reaction by using the kit provided by the invention, the defect that an existing kit cannot simultaneously detect the salmonella enteritidis and the mycoplasma synoviae is effectively overcome, and the kit is suitable for large-scale popularization and application of the salmonella enteritidis and the mycoplasma synoviae. Powerful technical support is provided for purification of chicken enteritis salmonellosis and mycoplasma synoviae in chicken farms, and the method has high practical value and wide application prospect.

Description

technical field [0001] The invention relates to the technical field of animal pathogen detection, in particular to a dual fluorescent PCR primer set, a kit and a method for detecting Salmonella enteritidis and Mycoplasma synovialis. Background technique [0002] Mycoplasma synoviae (MS) is an important pathogen that threatens the healthy breeding of chickens, and can cause Mycoplasma synovialis disease in chickens. All breeds and day-old chickens can be infected with the disease. Chickens aged 1 to 4 months are most seriously affected. The incidence rate is generally 5% to 10%, and the fatality rate does not exceed 10%. The clinical manifestations are stunted growth and development of chickens, reduced feed conversion rate, bursitis, tenosynovitis, blisters on the chest keel, swollen paw pads, swollen hock joints, lameness or even unable to stand, poor eggshell quality, brittle, feeding The chicken industry has caused serious economic losses. Chickens can also carry the ba...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12N15/11C12R1/42C12R1/35
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2537/143C12Q2563/107C12Q2527/107Y02A50/30
Inventor 于新友王文秀李天芝谢金文李峰唐娜
Owner SHANDONG BINZHOU ANIMAL SCI & VETERINARY MEDICINE ACADEMY
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