CN115961094B - Dual PCR primer and kit for identifying African swine fever and streptococcus suis and application of dual PCR primer and kit - Google Patents
Dual PCR primer and kit for identifying African swine fever and streptococcus suis and application of dual PCR primer and kit Download PDFInfo
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Abstract
The invention discloses a double PCR primer and a kit for identifying African swine fever and streptococcus suis and application thereof. The double PCR primers for identifying the African swine fever and the streptococcus suis comprise an African swine fever upstream primer, an African swine fever downstream primer, an African swine fever probe, a streptococcus suis upstream primer, a streptococcus suis downstream primer and a streptococcus suis probe, and the nucleic acid sequences of the primers are shown as SEQ ID NO. 1-6. The primers and the probes can be used for identifying African swine fever and streptococcus suis at the same time, are favorable for inhibiting, preventing and controlling African swine fever virus and streptococcus suis, reduce economic loss, and have the advantages of simple whole detection process, short detection time and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of PCR detection, and particularly relates to a double PCR primer and a kit for identifying African swine fever and streptococcus suis and application thereof.
Background
African swine fever (African Swine fever, ASF) is an acute, highly contagious infectious disease of domestic pigs and wild pigs caused by African swine fever virus (African Swine fevervirus, ASFV), is clinically characterized by hyperpyrexia, anorexia, bleeding of skin and internal organs, and generally dies 2-10 days after the illness, and the death rate can reach 100% in severe cases. Streptococcus suis (Streptococcus Suis, SS) is a national regulation of a second type of animal epidemic disease, which is an acute and febrile infectious disease commonly suffered by humans and animals, is a main pathogen causing streptococcus suis, and mainly causes epidemic diseases such as meningitis, septicemia and the like of pigs.
African swine fever and streptococcus suis are similar in clinical symptoms, high fever and septicemia and similar in epidemic characteristics, so that the African swine fever and streptococcus suis are often confused, but pathogenesis and treatment means of the African swine fever and streptococcus suis are different. At present, african swine fever and streptococcus suis infection often occur in a mixed mode, clinical manifestations and pathological changes are complex and changeable, the disease is difficult to distinguish and judge, the disease is urgent, the disease is short in course and high in death rate, the disease becomes a main factor which seriously affects the sustainable development of regional pig farming industry health class, and the characteristics of similar clinical symptoms and similar epidemic characteristics bring certain difficulty to clinical diagnosis, so that the optimal prevention and treatment period is often delayed, and great economic loss is caused to the farming industry. Therefore, high importance is required for the two epidemic diseases, and the periodical quarantine is important to prevent the occurrence and the spread of the diseases and reduce the loss of the diseases to the pig industry.
The method for identifying the African swine fever and the streptococcus suis has the problems of long time consumption and low accuracy according to epidemiological investigation, clinical symptoms, pathological anatomy change technology and the like, and PCR is used as a novel molecular biology technology, and since birth, a large number of target fragments can be obtained in a short time due to high specificity and sensitivity, so that the PCR becomes one of the important means of the current invention research. However, there is currently a lack of differential diagnostic kits for these two pathogens (African swine fever virus and Streptococcus suis).
Disclosure of Invention
Aiming at the problem of difficult simultaneous identification of African swine fever and streptococcus suis in the prior art, the invention provides a double PCR primer and a kit for identifying the African swine fever and the streptococcus suis and application thereof, and the African swine fever virus and the streptococcus suis can be detected simultaneously.
The invention is realized by the following technical scheme:
a dual PCR primer for identifying african swine fever and streptococcus suis, comprising an african swine fever upstream primer, an african swine fever downstream primer, a streptococcus suis upstream primer, a streptococcus suis downstream primer:
the African swine fever upstream primer sequence is shown as SEQ ID NO. 1;
the African swine fever downstream primer sequence is shown as SEQ ID NO. 2;
the upstream primer sequence of streptococcus suis is shown as SEQ ID NO. 4;
the upstream primer sequence of streptococcus suis is shown as SEQ ID No. 5.
The invention discloses a double PCR primer kit for identifying African swine fever and streptococcus suis, which comprises an African swine fever upstream primer, an African swine fever downstream primer, a streptococcus suis upstream primer and a streptococcus suis downstream primer:
the African swine fever upstream primer sequence is shown as SEQ ID NO. 1;
the African swine fever downstream primer sequence is shown as SEQ ID NO. 2;
the upstream primer sequence of streptococcus suis is shown as SEQ ID NO. 4;
the upstream primer sequence of streptococcus suis is shown as SEQ ID No. 5.
Further, the double PCR primer kit for identifying African swine fever and streptococcus suis also comprises an African swine fever probe and a streptococcus suis probe, wherein the African swine fever probe has a sequence shown as SEQ ID NO. 3, and the streptococcus suis probe has a sequence shown as SEQ ID NO. 6.
Further, the dual PCR primer kit for identifying African swine fever and streptococcus suis also comprises 2 Xmix PCR buffer solution and ddH2O.
In the invention, the double PCR primer kit for identifying the African swine fever and the streptococcus suis is applied to identifying the African swine fever virus and the streptococcus suis.
Further, the composition of the PCR reaction system is as follows: 10 mu L of 2 XmixPCR buffer, 0.5 mu L of African swine fever upstream primer and 10 mu M; african swine fever downstream primer 0.5 μL, 10 μM, streptococcus suis upstream primer 0.5 μL, 10 μM, streptococcus suis downstream primer 0.5 μL, 10 μM, african swine fever probe 0.5 μL, 10 μM, streptococcus suis probe 0.5 μL, 10 μM, ddH2O 6.0 μL, and template 6.0 μL.
Further, the PCR reaction procedure was: in the first stage, 50 ℃ for 2min; in the second stage, 95 ℃ for 3min; in the third stage, fluorescence was collected at 95℃for 10sec,60℃for 30sec,45 cycles, and at 60℃for each cycle of annealing extension in the third stage.
Advantageous effects
The invention utilizes the double PCR primers of African swine fever and streptococcus suis to identify the African swine fever and streptococcus suis, can simultaneously distinguish the African swine fever virus and the streptococcus suis, is beneficial to inhibiting, preventing and controlling the African swine fever virus and the streptococcus suis, reduces economic loss, and has simple whole detection process, short detection time and high sensitivity.
Drawings
FIG. 1 is a graph of gene-specific amplification of African Swine Fever (ASFV) virus, wherein blue is FAM channel, a is ASFV sample, b is ASFV positive control, and C is ASFV negative control; green is VIC channel, d represents no result;
FIG. 2 is a graph showing gene-specific amplification of Streptococcus Suis (SS), wherein a is Streptococcus suis 1, b is Streptococcus suis 2, c is Streptococcus agalactiae, streptococcus polysaccharideus, streptococcus mammary, streptococcus agalactiae;
FIG. 3 is a graph showing amplification curves of Streptococcus Suis (SS) genes at various concentrations;
FIG. 4 is a graph showing dual specificity amplification curves of African Swine Fever (ASFV) virus genes and Streptococcus Suis (SS) genes, wherein blue is FAM channel, a is ASFV sample, b is ASFV positive control, and C is ASFV negative control; green is VIC channel, d is SS sample; e is SS positive control and f is SS negative control.
Detailed Description
For a better understanding of the present invention, the technical solutions described in the present invention will be further described with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
(1) Selection and design of African swine fever ASFV primer probe
Based on the p30, p54 and p72 sequences of NCBI, comparing the conservation of the p30, p54 and p72 gene sequences, determining p70 as a target gene by screening, and amplifying the target gene with 141bp, wherein the final African swine fever upstream primer, the African swine fever downstream primer and the African swine fever probe are designed according to SEQ ID NOs shown in the following table 1: 1-3, both primers and probes were synthesized by Shanghai Biotechnology Co., ltd (the same applies below).
(2) Selection and design of streptococcus suis SS (Streptococcus suis) 1-34 universal primer probes
Comparing the sequence conservation of recX, 16srRNA and GroEL genes according to the sequence of recX, 16srRNA and GroEL downloaded by NCBI, and determining GroEL as a target gene by screening; then downloading GroEL gene fragment complete sequences of streptococcus suis types 1-34 from NCBI, and designing primers and probes; the final streptococcus suis upstream primer, streptococcus suis downstream primer and streptococcus suis probe were designed as set forth in SEQ ID NOs: 4-6.
TABLE 1 African swine fever and Streptococcus suis primer probe sequences
Example 2
Establishing an African swine fever fluorescence PCR detection method;
african swine fever probe [ FAM ] -GCGTGTAAACGGCGCCCTCT- [ BHQ1]
(1) Fluorescent PCR reaction system
The 2 Xmix PCR buffer, ddH 2 O was purchased from Vazyme, inc., and the above Template was obtained by extraction from a viral DNA/RNA extraction kit (4.0) (Siami Tianlong technologies Co., ltd.).
(2) Fluorescent PCR reaction conditions
The first stage: 50 ℃ for 2min;
and a second stage: 3min at 95 ℃;
and a third stage: fluorescence was collected at 60℃for each cycle of the third stage at 95℃for 10sec,60℃for 30sec,45 cycles.
(3) Analytical detection was performed using a multichannel PCR apparatus, and the results were read using FAM (465-510) and VIC (533-580) channels, respectively, and the African swine fever fluorescence PCR amplification results were shown in FIG. 1, in which blue: FAM channels; a, ASFV sample; b, ASFV positive control; ASFV negative control; green: a VIC channel; d: without result.
Example 3
The fluorescence PCR detection method of streptococcus suis is established;
streptococcus suis probe [ VIC-TCGGTGCCGCTACTGAGACTGA- [ BHQ2]
(1) Fluorescent PCR reaction system
The 2 Xmix PCR buffer, ddH 2 O was purchased from Vazyme, inc., and the above Template was obtained by extraction from a bacterial genomic DNA miniprep kit (TAKARA, inc.).
(2) Fluorescent PCR reaction conditions
The first stage: 95 ℃ for 10min;
and a second stage: fluorescence was collected at 95℃for 15sec,60℃for 1min,40 cycles, and at 60℃for each cycle of the second stage for annealing extension.
(3) Nucleic acids of 6 clinical samples (two strains of Streptococcus suis, the remaining four strains being non-Lactobacillus deltoid Streptococcus, multiple animal Streptococcus, streptococcus mammary, streptococcus agalactiae) were extracted with a bacterial genome extraction kit, and their genomes were diluted 10-fold as templates, and the PCR amplification results are shown in FIG. 2, wherein a is Streptococcus suis 1, b is Streptococcus suis 2, and c is non-Lactobacillus deltoid Streptococcus, multiple animal Streptococcus, streptococcus mammary, streptococcus agalactiae.
(4) The extracted streptococcus suis 1 template is serially diluted by 10 times, and fluorescent PCR reaction is carried out according to the conditions determined in the step (1) and the step (2), and the PCR amplification result is shown in the following figure 3, wherein the a-d concentration is sequentially reduced.
Example 4
Establishment of African swine fever and streptococcus suis dual fluorescence PCR detection method
The fluorescence PCR detection method of streptococcus suis is established;
(1) Fluorescent PCR reaction system
The 2 Xmix PCR buffer, ddH2O, was purchased from Vazyme.
(2) Fluorescent PCR reaction conditions
The first stage: 50 ℃ for 2min;
and a second stage: 3min at 95 ℃;
and a third stage: fluorescence was collected at 60℃for each cycle of the third stage at 95℃for 10sec,60℃for 30sec,45 cycles.
(3) Detecting by adopting a multichannel PCR instrument, and respectively reading results by using FAM (465-510) channels and VIC (533-580) channels, wherein blue is FAM channels, a is an ASFV sample, b is an ASFV positive control, and C is an ASFV negative control; green is VIC channel, d is SS sample; e is SS positive control and f is SS negative control.
(4) African swine fever virus and streptococcus suis results identification:
1) Negative: both detection channels have no Ct/Cp value and no characteristic amplification curve, which indicates that the sample has no African swine fever virus and streptococcus suis;
2) Single positive: if only the FAM detection channel has a characteristic amplification curve, the Cp/Ct value is less than or equal to 42.0, and the VIC detection channel has no Cp/Ct value and no amplification curve, the sample only contains African swine fever virus; if only the VIC detection channel has a characteristic amplification curve, the Cp/Ct value is less than or equal to 42.0, and the FAM detection channel has no Cp/Ct value and no amplification curve, the fact that only streptococcus suis exists in the sample is indicated;
3) Double positive: for example, typical amplification curves appear in FAM detection channels and VIC detection channels, and Ct/Cp values are less than or equal to 42.0, which indicates that African swine fever virus and streptococcus suis exist in the sample.
Claims (6)
1. A dual PCR primer probe combination for identifying african swine fever and streptococcus suis, comprising an african swine fever upstream primer, an african swine fever downstream primer, an african swine fever probe, a streptococcus suis upstream primer, a streptococcus suis downstream primer, and a streptococcus suis probe:
the African swine fever upstream primer sequence is shown as SEQ ID NO. 1;
the African swine fever downstream primer sequence is shown as SEQ ID NO. 2;
the African swine fever probe sequence is shown as SEQ ID NO. 3;
the upstream primer sequence of streptococcus suis is shown as SEQ ID NO. 4;
the upstream primer sequence of streptococcus suis is shown as SEQ ID NO. 5;
the streptococcus suis probe sequence is shown as SEQ ID NO. 6.
2. The double PCR kit for identifying African swine fever and streptococcus suis is characterized by comprising a double PCR primer probe combination for identifying African swine fever and streptococcus suis, wherein the double PCR primer probe combination for identifying African swine fever and streptococcus suis comprises an African swine fever upstream primer, an African swine fever downstream primer, an African swine fever probe, a streptococcus suis upstream primer, a streptococcus suis downstream primer and a streptococcus suis probe:
the African swine fever upstream primer sequence is shown as SEQ ID NO. 1;
the African swine fever downstream primer sequence is shown as SEQ ID NO. 2;
the African swine fever probe sequence is shown as SEQ ID NO. 3;
the upstream primer sequence of streptococcus suis is shown as SEQ ID NO. 4;
the upstream primer sequence of streptococcus suis is shown as SEQ ID NO. 5;
the streptococcus suis probe sequence is shown as SEQ ID NO. 6.
3. The dual PCR kit for identifying African swine fever and streptococcus suis as recited in claim 2, further comprising 2 Xmix PCR buffer and ddH 2 O。
4. Use of a dual PCR kit for the identification of african swine fever and streptococcus suis according to claim 2 or 3, for the identification of african swine fever virus and streptococcus suis, wherein the use is for non-disease diagnostic purposes.
5. According to claim 4The application is characterized in that the composition of a PCR reaction system is as follows: 10 mu L of 2 XmixPCR buffer, 0.5 mu L of African swine fever upstream primer and 10 mu M; african swine fever downstream primer 0.5 mu L, 10 mu M, streptococcus suis upstream primer 0.5 mu L, 10 mu M, streptococcus suis downstream primer 0.5 mu L, 10 mu M, african swine fever probe 0.5 mu L, 10 mu M, streptococcus suis probe 0.5 mu L, 10 mu M, ddH 2 O6.0. Mu.L, template 6.0. Mu.L.
6. The use according to claim 4, wherein the PCR reaction procedure is: in the first stage, 50 ℃ for 2min; in the second stage, 95 ℃ for 3min; in the third stage, fluorescence was collected at 95℃for 10sec,60℃for 30sec,45 cycles, and at 60℃for each cycle of annealing extension in the third stage.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110760620A (en) * | 2019-12-12 | 2020-02-07 | 黑龙江八一农垦大学 | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method |
| CN111286559A (en) * | 2020-03-06 | 2020-06-16 | 广东海大畜牧兽医研究院有限公司 | Primer, probe and kit for detecting African swine fever virus |
| WO2022136624A1 (en) * | 2020-12-24 | 2022-06-30 | Intervet International B.V. | African swine fever diva immunoassay |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110760620A (en) * | 2019-12-12 | 2020-02-07 | 黑龙江八一农垦大学 | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method |
| CN111286559A (en) * | 2020-03-06 | 2020-06-16 | 广东海大畜牧兽医研究院有限公司 | Primer, probe and kit for detecting African swine fever virus |
| WO2022136624A1 (en) * | 2020-12-24 | 2022-06-30 | Intervet International B.V. | African swine fever diva immunoassay |
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