CN110904251B - Primer group and kit for detecting duck mycoplasma - Google Patents
Primer group and kit for detecting duck mycoplasma Download PDFInfo
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- CN110904251B CN110904251B CN201911192095.0A CN201911192095A CN110904251B CN 110904251 B CN110904251 B CN 110904251B CN 201911192095 A CN201911192095 A CN 201911192095A CN 110904251 B CN110904251 B CN 110904251B
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Abstract
The invention relates to a primer group and a kit for detecting a duck mycoplasma, belonging to the technical field of pathogen detection. The primer group for detecting the duck mycoplasma comprises primers shown as SEQ ID NO. 1 and SEQ ID NO. 2; the kit for detecting the duck mycoplasma comprises the primer group. The invention provides a specific primer aiming at a duck mycoplasma, and the specific primer can be used for specifically detecting the duck mycoplasma; the kit can detect the duck mycoplasma by adopting PCR amplification, so that the invention provides a method for quickly and specifically detecting the duck mycoplasma, and provides a powerful technical means for clinically controlling the infection of the duck mycoplasma; the total detection time of the kit is about 2.5h, the kit is easy to operate, convenient and quick, and the aim of quick detection can be fulfilled.
Description
Technical Field
The invention relates to a primer group and a kit for detecting a duck mycoplasma, belonging to the technical field of pathogen detection.
Background
Duck Mycoplasma (MA), also called Mycoplasma, causes chronic respiratory diseases, called "slow breathing" for short, mainly affects respiratory tract, is characterized by slow development, long course of disease, can infect ducks of different ages of day, and is most susceptible for 1-2 weeks; the disease spreads in duck groups for a long time, especially in spring with changeable climate, mainly through respiratory tract, digestive tract and hatching eggs; the disease incidence is high, after the disease occurs, the resistance of the duck body is reduced, colibacillosis is easy to be complicated, if the colibacillosis is complicated, a large number of ducks die, and great economic loss is caused to the duck breeding industry.
The detection method of mycoplasma mainly comprises pathogen separation culture, serological method and molecular biological method. The molecular biology method is a detection method which is applied more generally, but a PCR detection method for identifying and diagnosing the mycoplasma anatis does not exist at present. The existing detection method for mycoplasma has long clinical detection period and high cost, and can not quickly diagnose the mycoplasma anatis.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the primer group and the kit for detecting the duck mycoplasma.
In order to achieve the purpose, the invention adopts the technical scheme that:
in a first aspect, the invention provides a primer group for detecting mycoplasma anatipestifer, which comprises primers shown as SEQ ID NO. 1 and SEQ ID NO. 2.
The duck Mycoplasma (MA) causes chronic respiratory diseases of ducks, takes inflammation of infraorbital sinuses as a main characteristic, influences the growth of ducklings and adult ducks, causes low feed conversion rate, remarkably reduces egg yield and causes serious economic loss for duck breeding households. The invention selects 16s rRNA of a Mycoplasma anatum 1340 model strain (NR _ 025176.1) as a target gene, selects a specific region of a target gene sequence through the comparison analysis of the gene sequence, and designs and synthesizes primers shown in SEQ ID NO 1 and SEQ ID NO 2. The size of the amplified band of the primer group is 550bp.
When primers are designed for the specific region of the 16s rRNA target gene, numerous primers with different lengths, starting points and end points can be designed. The inventor researches the availability of each primer, continuously adjusts the design parameters of the primers, and finally selects the primers of the invention through a large amount of screening. The primer has few hairpin structures, low mismatching rate and good amplification specificity. The primer has good specificity when being used for detecting the duck mycoplasma.
In a second aspect, the invention provides an application of the primer group in preparation of a kit for detecting the mycoplasma anatipestifer.
In a third aspect, the invention provides a kit for detecting a duck mycoplasma, which comprises the primer group.
As a preferred embodiment of the kit for detecting the mycoplasma anatis, the kit further comprises a PCR premix and water. The kit for detecting the duck mycoplasma is a PCR detection kit, and the kit for detecting the duck mycoplasma has the advantages of high sensitivity, good specificity, high stability, visual result, easiness in operation, convenience and quickness, capability of greatly shortening the detection time and powerful technical means for clinically controlling the infection of the duck mycoplasma.
As a preferred embodiment of the kit for detecting the mycoplasma anatis, the PCR premix comprises DNA polymerase, dNTPS and MgCl 2 And PCR buffer.
As a preferred embodiment of the kit for detecting the mycoplasma anatis, the kit further comprises a DNA extraction reagent.
As a preferred embodiment of the kit for detecting the mycoplasma anatis, the DNA extraction reagent is PBS buffer solution. The PBS buffer solution can be used for extracting mycoplasma from lung, ovary and semen of the duck.
As a preferred embodiment of the kit for detecting the mycoplasma anatis, the kit further comprises a negative control substance and a positive control substance.
As a preferable embodiment of the kit for detecting the duck mycoplasma, the negative control substance is double distilled water, and the positive control substance comprises a duck mycoplasma genome.
As a preferable embodiment of the kit for detecting the duck mycoplasma, the sample for detecting the kit is a tissue disease sample of a duck, a nasopharyngeal swabbing sample or semen. Preferably, the sample for detection by the kit is lung, ovary or semen of duck.
The use method of the kit comprises the following steps:
(1) Extracting DNA in a sample to be detected by adopting a DNA extraction reagent;
(2) Mixing the DNA extracted in the step (1) with the primer group and the PCR premixed solution to prepare a PCR reaction solution by taking the DNA as a template, and performing PCR amplification;
(3) And (3) respectively carrying out electrophoresis detection on the products amplified by the PCR in the step (2), the negative control product amplification products and the positive control product amplification products through agarose gel electrophoresis, and judging.
The standard for judging whether the mycoplasma anatis in the sample to be detected in the step (3) is as follows: the negative control has no amplified band, the amplified band size of the positive control is 550bp, which indicates that the control is established; a 550bp strip appears in a PCR amplification product of a sample to be detected, and the sample is infected by the duck mycoplasma; the PCR amplification product of the sample to be detected has no band, and the result is negative.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention provides a specific primer aiming at a duck mycoplasma, the specific primer can be used for specifically detecting the duck mycoplasma, and the amplification band size of the specific primer is 550bp.
(2) The kit can detect the duck mycoplasma by adopting PCR amplification, thereby providing a method for quickly and specifically detecting the duck mycoplasma and providing a powerful technical means for clinically controlling the infection of the duck mycoplasma. The total detection time of the kit is about 2.5h, the kit is easy to operate, convenient and quick, and the aim of quick detection can be fulfilled. The invention is suitable for rapid detection of duck farms and also suitable for epidemiological investigation.
(3) The detection limit of the invention is 1 pg/mu L, which shows that the PCR detection method of the mycoplasma anatis has high sensitivity, and the detection method of the invention can shorten the detection time and reduce the use cost.
Drawings
FIG. 1 is a specific test electrophoretogram of the PCR detection method for Mycoplasma anatis disclosed;
FIG. 2 is a sensitivity test electrophoretogram of the PCR detection method for Mycoplasma anatum of the present invention;
FIG. 3 shows the result of the electrophoresis test of a part of clinical samples by the PCR test method for Mycoplasma anatum of the present invention.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
Example 1
In an embodiment of the primer set for detecting the mycoplasma hyopneumoniae, the primer set for detecting the mycoplasma hyopneumoniae comprises an upstream primer MA-F and a downstream primer MA-R. The base sequence of the upstream primer MA-F is shown as SEQ ID NO. 1, and the base sequence of the downstream primer MA-R is shown as SEQ ID NO. 2. See in particular table 1 below.
TABLE 1
The kit for detecting the mycoplasma anatis comprises 2 xtap PCR Mix (the PCR Mix comprises DNA polymerase, dNTPS and MgCl) 2 And PCR buffer solution), sterilized double distilled water (used for supplementing the balance of a PCR reaction system), the MA upstream primer and the MA downstream primer, a negative control substance and a positive control substance, wherein the negative control substance is sterilized double distilled water, and the positive control substance is a Mycoplasma anatum genome. The kit of this embodiment may further comprise a DNA extraction reagent, preferably, the DNA extraction reagent is PBS buffer.
Example 2
(1) Design of PCR primers
Selecting 16s rRNA of a Mycoplasma anatum 1340 model strain (NR _ 025176.1) as a target gene, carrying out BLAST comparison analysis on the target gene, selecting a difference region of a target gene sequence to design and synthesize a pair of Mycoplasma anatum specific amplification primers, paying attention to that a difference site is located at the 3' end of the primers as much as possible in the primer design process, and simultaneously adjusting primer parameters to reduce a hairpin structure, reduce the mismatch rate and ensure the amplification specificity. The primers were then PCR verified to determine primer availability, including specificity and sensitivity assessments. The Mycoplasma anatis primer information is shown in Table 1. The above primers were synthesized by Biotechnology engineering (Shanghai) Co., ltd.
(2) Specificity verification
The mycoplasma anatipestifer 5 strains (Y1, Y2, Y07-F5, 16-F3, F3-F6) are provided by the university of Huazhong agriculture microbial and immunological laboratory, the mycoplasma synoviae attenuated vaccine strain F, the mycoplasma gallisepticum virulent strain S6, the HS strain, the salmonella (CVCC 542) and the Escherichia coli (ATCC 25922) are provided by the university of Huazhong agriculture microbial and immunological laboratory, the mycoplasma synoviae (WVU 1853) is provided by the university of Gansu agriculture preventive veterinary system, and the streptococcus (SC 19) is provided by the university of Huazhong agriculture preventive veterinary system agricultural microbial national focus laboratory.
The above samples were used for specificity evaluation, and total nucleic acid extraction was performed separately, and the total nucleic acid extracted from each sample was dissolved in TE buffer at a concentration of 10 ng/. Mu.L.
Using the total nucleic acid of each sample obtained above as a template, PCR amplification was performed using the primer set obtained above, and a negative control was set.
The reaction system was prepared as follows: 2 × Tap PCR Mix10 μ L, upstream and downstream primers 1 μ L each, DNA template 1 μ L, ultrapure water to make up to 20 μ L.
The reaction conditions include the following steps a-f: a: 5min at 95 ℃; b: 30s at 95 ℃; c, at 54 ℃ for 30s; d: 50s at 72 ℃; b-d 34 reactions were cycled; e: 7min at 72 ℃; f: infinity at 4 ℃.
The amplification products were analyzed by nucleic acid gel, and the results are shown in FIG. 1. As can be seen from FIG. 1, the 550bp band can be amplified only by Mycoplasma anatum, and no band exists in other controls and negative controls, which indicates that the primer of the embodiment is difficult to be interfered by unrelated pathogens and has high specificity.
(3) Sensitivity verification
Adjusting the concentration of the Mycoplasma anatum DNA obtained in the step (2) to 10 mug// muL, diluting the Mycoplasma anatum DNA to 100 ng/muL according to 10-fold concentration gradient, preparing a PCR reaction system by using each concentration gradient genome as a template to perform PCR reaction, performing electrophoresis observation after the amplification is finished, and obtaining the test result shown in figure 2.
As can be seen from FIG. 2, the detection limit of the PCR detection method for Mycoplasma anatum of the present invention is 1pg/μ L, which indicates that the PCR detection method for Mycoplasma anatum of the present invention has high sensitivity.
(4) Field test
40 parts of semen and tissue samples suspected of causing the duck mycoplasma disease are collected, and the pathogen infection condition of the duck mycoplasma is detected by the detection method of the duck mycoplasma obtained in the embodiment 1, which comprises the following steps:
example 3
(1) Processing of tissue pathology samples
Collecting suspected pathological tissue pathological material such as lung, ovary and semen, cutting 3-5g pathological material into paste, adding 5 times sterile PBS buffer solution (same operation as semen), grinding thoroughly with tissue grinder, collecting suspension, centrifuging at 4 deg.C at 10000r/min for 10min, and sucking supernatant.
(2) DNA extraction
And taking 200 mu L of supernatant, carrying out water bath at 100 ℃ for 15min, immediately carrying out ice bath for 10min, and centrifuging at 12000r/min for 10min to obtain the supernatant as the template DNA. The negative control adopts sterilized double distilled water.
(3) PCR amplification reaction
Taking the extracted sample DNA as a template, and preparing a reaction system according to the following operations: 2 × Tap PCR Mix10 μ L, upstream and downstream primers 1 μ L each, DNA template 1 μ L, ultrapure water to make up to 20 μ L. Negative (sterilized double distilled water) and positive (duck mycoplasma genome) controls are set at the same time.
Carrying out a PCR amplification reaction under conditions comprising the steps of: a: performing pre-denaturation reaction at 95 ℃ for 5min; b: reacting at 95 ℃ for 30s; c, reacting at 54 ℃ for 30s; d: reacting at 72 ℃ for 50s; b-d, 34 circulation reactions are circulated; e: reacting at 72 ℃ for 7min; f: infinity at 4 ℃.
(4) Electrophoretic detection
Detecting all PCR amplification products through 1% agarose gel electrophoresis, taking DL2000 molecular mass standard as reference, wherein a negative control does not generate a band, and a positive control amplifies a band with the size of 550bp to indicate that the control is true; a 550bp strip appears in a PCR amplification product of a sample to be detected, and the sample is the duck mycoplasma infection; and if the PCR amplification product of the sample to be detected has no band, the sample to be detected is negative.
The results of the electrophoretic detection in example 3 were: 30 samples of 40 clinical samples are semen, and the positive rate is 90%;10 parts of the test sample are lung tissue samples, and the positive rate is 40%. FIG. 3 shows the results of the detection of a portion of the samples, and it can be seen from FIG. 3 that the detection results of the samples in lane 10 are all positive for Mycoplasma anatis infection. The PCR detection method for the mycoplasma anatipestifer has good practicability and applicability.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Win food group Ltd
<120> primer group and kit for detecting duck mycoplasma
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213> Artificial sequence
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cacgtactta acataccctt t 21
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence
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ttcataactc tagttcgcca g 21
Claims (5)
1. A kit for detecting a duck mycoplasma is characterized by comprising primers shown as SEQ ID NO. 1 and SEQ ID NO. 2, PCR premixed solution, water, a DNA extraction reagent, a negative control substance and a positive control substance.
2. The kit for detecting a Mycoplasma anatis of claim 1, wherein said PCR premix comprises DNA polymerase, dNTPS, mgCl 2 And PCR buffer.
3. The kit for detecting a Mycoplasma anatis of claim 1, wherein said DNA extraction reagent is PBS buffer.
4. The kit for detecting the mycoplasma hyopneumoniae according to claim 1, wherein the negative control substance is double distilled water, and the positive control substance comprises a mycoplasma hyopneumoniae genome.
5. The kit for detecting a mycoplasma hyorhinis according to claim 1, wherein the sample for detection by the kit is a tissue morbid sample of duck, a nasopharyngeal swabbing sample or semen.
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| KR20150092834A (en) * | 2014-02-06 | 2015-08-17 | 원광대학교산학협력단 | Primer set for the multidetection of mycoplasma genus, method for the multidetection of mycoplasma genus by using the primer, and kit for the multidetection of mycoplasma genus |
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| CN101481742B (en) * | 2009-02-11 | 2010-12-15 | 中国农业大学 | Detection kit for Mycoplasma hyopneumoniae and use thereof |
| KR101279395B1 (en) * | 2011-08-22 | 2013-06-27 | 원광대학교산학협력단 | Primer set for the detection of Mycoplasma hyorhinis, kit for the detection of Mycoplasma hyorhinis by using the primer set, and kit for the diagnosis of porcine pneumonia by using the primer set |
| CN103937892B (en) * | 2014-04-21 | 2015-07-29 | 华南农业大学 | The multiple PCR detection primer group of duck source various pathogens and test kit thereof |
| CN105462884B (en) * | 2015-12-18 | 2018-12-04 | 中国农业科学院兰州兽医研究所 | The isolation and purification method of mycoplasma anatis culture medium and mycoplasma anatis |
| WO2017178587A1 (en) * | 2016-04-14 | 2017-10-19 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Method for universal detection and quantification of mycoplasma (mollicutes) 16s rdna by quantitative polymerase chain reaction amplifying a 1.5 kilobase fragment |
| CN107254534A (en) * | 2017-07-05 | 2017-10-17 | 中国农业科学院兰州兽医研究所 | A kind of kit of TaqMan probe fluorescence quantitative PCR detection mycoplasma anatis |
| CN109825648B (en) * | 2019-04-02 | 2023-02-28 | 江苏省农业科学院 | Primer composition and kit for detecting mycoplasma ovipneumoniae and peste des petits ruminants virus and application |
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| KR20150092834A (en) * | 2014-02-06 | 2015-08-17 | 원광대학교산학협력단 | Primer set for the multidetection of mycoplasma genus, method for the multidetection of mycoplasma genus by using the primer, and kit for the multidetection of mycoplasma genus |
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| Title |
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| 鸭支原体病并发大肠杆菌病诊疗实例;邢兰君;《新农业》;20121210(第17期);第37页 * |
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