CN115976234A - Streptococcus bovis primer group based on 16s rRNA and LAMP detection method thereof - Google Patents
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Abstract
The invention provides a streptococcus bovis primer group based on 16s rRNA and an LAMP detection method thereof, and relates to the technical field of rapid detection of molecular diagnosis. The primer group comprises primers F3-3, B3-3, FIP-3, BIP-3, FL-3 and BL-3; the nucleotide sequences of the primers F3-3, B3-3, FIP-3, BIP-3, FL-3 and BL-3 are respectively shown as SEQ ID No. 13-SEQ ID No. 18; the LAMP detection method comprises the detection reagent, reaction conditions and a primer group. The LAMP detection method for the specific streptococcus bovis, which is established by using the primers designed by the conserved sequence 16s rRNA of the streptococcus bovis, can quickly complete specific amplification within 30-60 minutes without a special PCR amplification instrument on a detection field, and the result is visible to the naked eye, so that the LAMP detection method has the characteristics of high sensitivity, strong specificity and no need of special equipment, can realize real-time quick detection of the streptococcus bovis infection in application scenes such as pastures, farms, slaughterhouses, food factories, environments and the like, is favorable for quickly diagnosing bovine diseases at present in time and quickly identifying the streptococcus bovis, and provides a new molecular diagnosis combination for treatment of infectious diseases caused by the streptococcus bovis and prevention and control of epidemic situations of the streptococcus bovis.
Description
Technical Field
The invention relates to the technical field of rapid detection of molecular diagnosis, in particular to a streptococcus bovis primer group based on 16s rRNA and an LAMP detection method thereof.
Background
Bovine Streptococcus (Streptococcus bovis) is a common facultative anaerobe in the rumen, belongs to group D Streptococcus, and includes 4 different species, namely Streptococcus equi (Streptococcus equinus), streptococcus gallyticus (Streptococcus galllyticus), streptococcus infantis (Streptococcus infantis) and Streptococcus agalactiae (Streptococcus alactopterius). McNeal and Blevins reported for the first time in 1945 that S.bovis can cause infective endocarditis, and there was a correlation between S.bovis endocarditis and colon cancer. Streptococcus bovis is not only a gastrointestinal resident bacterium, but also a serious pathogenic bacterium, and can cause septicemia and endocarditis. The gastrointestinal tract is a common invasion route of the bacterium, and the urinary tract, the liver and gall system and the like are possible invasion routes. Bovine streptococcus can cause mutual infection of human and animals, and brings huge economic loss to the breeding industry, so that the harmfulness of streptococcus is highly valued, the health of human beings and the food safety are guaranteed, the prevalence and the spread of bovine streptococcosis are effectively controlled, and the health and the sustainable development of the breeding industry are guaranteed.
At present, the detection of the streptococcus bovis mainly depends on bacteria isolation culture and a 16s rRNA sequencing technology, but the isolation culture of the bacteria is complex and time-consuming, and needs to be operated by professional technical personnel in a professional laboratory, so the sequencing technology can be completed only by professional sequencing companies, and the rapid field detection of livestock infected with the streptococcus bovis cannot be realized in an animal farm. Loop-mediated isothermal amplification (LAMP) technology couples a target gene with 4 to 6 primers, utilizes the strand displacement activity of Bst DNA polymerase to perform efficient amplification under a constant temperature condition, can judge an amplification result by observing a fluorescent amplification curve or adding indicators such as nucleic acid dyes and the like, has the characteristics of high sensitivity, strong specificity, no need of special equipment and the like, and can be applied to detection of pathogenic microorganisms.
Disclosure of Invention
Aiming at the problems, the invention designs a primer group by using the conserved sequence 16s rRNA of the streptococcus bovis, thereby improving the sensitivity and specificity of the streptococcus bovis detection; the LAMP detection method established by using the streptococcus bovis primer group has the advantages of no need of special equipment, short time and easy judgment of results through naked eyes, can realize real-time detection of streptococcus bovis infection in farms, animal farms and other places, is favorable for timely treatment of infected animals, and reduces economic loss.
The invention adopts the following technical scheme:
the invention provides a streptococcus bovis primer group based on 16s rRNA, which is designed by utilizing a streptococcus bovis conserved sequence 16s rRNA, and comprises primers F3-3, B3-3, FIP-3, BIP-3, FL-3 and BL-3; the nucleotide sequences of the primers F3-3, B3-3, FIP-3, BIP-3, FL-3 and BL-3 are respectively shown as SEQ ID No. 13-SEQ ID No. 18; the primer group has high specificity and sensitivity to streptococcus bovis.
The invention provides a LAMP detection method of streptococcus bovis based on 16s rRNA, which comprises the step of carrying out LAMP detection on the streptococcus bovis by using a detection reagent, an amplification reaction condition and the primer group.
Further, the detection reagent comprises 1.4mmol/L dNTP Mix, 20mmol/L Tris-HCl, 10mmol/L KCl and 10mmol/L (NH) 4 ) 2 SO 4 、6mmol/LMgSO 4 0.1% Triton X-100, 320U/mL Bst DNA polymerase, 10. Mu. Mol/L acid-base indicator.
Further, the acid-base indicator is selected from any one of phenol red, bromothymol blue, neutral red, phenolsulfonphthalein, cresol red and rosolic acid.
Further, the acid-base indicator is phenol red, and if the detection reagent changes from red to orange yellow, the detection reagent is positive for streptococcus bovis; negative and positive are not obvious when observed by naked eyes when hydroxynaphthol blue is used as an indicator; when the calcein is used as an indicator, the calcein needs to be added after reaction, so that the calcein is inconvenient to apply; and phenol red is obviously discolored as an indicator and has no influence on the reaction.
Further, the concentration of the primers F3-3, B3-3, FIP-3, BIP-3, FL-3 and BL-3 is 0.2. Mu. Mol/L, 1.6. Mu. Mol/L, 0.8. Mu. Mol/L and 0.8. Mu. Mol/L respectively.
Further, the amplification reaction time is 30-60min, and the temperature is 60-67 ℃.
Further, the optimal amplification reaction temperature is 65 ℃ and the time is 30min
Further, the detection limit of the LAMP method for detecting the 16s rRNA gene of the streptococcus bovis is 1.0 multiplied by 10 2 copies/μL。
A bovine streptococcus primer group based on 16s rRNA and an application of an LAMP detection method thereof in a bovine streptococcus detection kit.
The invention has the beneficial effects that:
the cattle streptococcus primer group provided by the invention is designed by taking 16s rRNA as a template, and has the characteristic of high specificity to cattle streptococcus; after LAMP amplification reaction, the color of the solution is changed, and whether the sample contains the streptococcus bovis can be judged by visual observation; the bovine streptococcus nucleic acid amplification method provided by the invention has low requirements on equipment, does not need large-scale instruments and equipment, and can be operated in the field, at home, on farms and the like; the operation method is simple, and complex training or professional technicians are not needed. The defects of long required time, large workload, complex operation and the like in the prior detection technology are overcome.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings of the embodiments will be briefly described below, and it is apparent that the drawings in the following description only relate to some embodiments of the present invention and are not limiting on the present invention.
FIG. 1 shows the results of screening Streptococcus bovis 16s rRNA gene primer combinations 1-5;
FIG. 2 shows the result of specificity of primer combination 1 for Streptococcus bovis 16s rRNA gene;
FIG. 3 shows the results of visualized screening of Streptococcus bovis, 16s rRNA gene temperature;
FIG. 4 is the result of agarose gel electrophoresis to verify the visualization screening of the 16s rRNA gene temperature of Streptococcus bovis;
FIG. 5 is the temperature screening amplification curve of the 16s rRNA gene of Streptococcus bovis;
FIG. 6 shows the results of visualization of the temperature screening of the 16s rRNA gene of Streptococcus bovis;
FIG. 7 shows the result of visualized detection of the 16s rRNA gene of Streptococcus bovis;
FIG. 8 is the result of visualizing the 16s rRNA gene of Streptococcus bovis by agarose gel electrophoresis;
FIG. 9 is a LAMP detection specific amplification curve of the 16s rRNA gene of Streptococcus bovis;
FIG. 10 shows LAMP detection specific amplification bands of Streptococcus bovis 16s rRNA gene;
FIG. 11 is a sensitive fluorescent amplification curve for LAMP method detection of Streptococcus bovis 16s rRNA gene;
FIG. 12 is a sensitive agarose electrophoresis strip for LAMP method detection of Streptococcus bovis 16s rRNA gene;
FIG. 13 is a visual detection result of the LAMP method for detecting the sensitivity of the 16s rRNA gene of Streptococcus bovis;
FIG. 14 is a linear regression analysis of the copy number of the standard plasmid of Streptococcus bovis 16s rRNA gene and LAMP fluorescent amplification curve.
Detailed Description
The invention is further illustrated below with reference to specific examples. It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
And (3) taking the streptococcus bovis DNA lysate sample as a positive control, and taking the DNA lysate not as a negative sample, and establishing the LAMP detection method. And (3) preparing the primer combination and the reaction system into a 25 mu L system, amplifying for 30-60min at the constant temperature of 65 ℃, and observing the result. And establishing a visual detection system by using an acid-base indicator phenol red, wherein the result is positive when the system is orange yellow through visual observation after the reaction is finished, and the result is negative when the system is red. And (3) establishing a fluorescent quantitative detection system by using SYBR Green I to perform quantitative verification on the established LAMP detection method, judging the detection result according to the fluorescent quantitative amplification curve, wherein the specific amplification curve is a positive result, and the amplification curve is a negative result. And (3) verifying the reliability of the detection result by using 2% agarose gel electrophoresis in the establishment process of the LAMP detection method.
Example design of primers for 16s rRNA of Streptococcus bovis
According to the 16s rRNA target gene of the streptococcus bovis, 5 sets of primer combinations are designed: combination 1 (F3-1, B3-1, FIP-1, BIP-1, FL-1 and BL-1, the sequences are shown as SEQ ID No. 1-SEQ ID No.6 respectively), combination 2 (F3-2, B3-2, FIP-2, BIP-2, FL-2 and BL-2, the sequences are shown as SEQ ID No. 7-SEQ ID No.12 respectively), combination 3 (F3-3, B3-3, FIP-3, BIP-3, FL-3 and BL-3, the sequences are shown as SEQ ID No. 13-SEQ ID No.18 respectively), combination 4 (F3-4, B3-4, FIP-4, BIP-4, FL-4 and BL-4, the sequences are shown as SEQ ID No. 19-SEQ ID No.24 respectively), and combination 5 (F3-5, B3-5, FIP-5, BIP-5, FL-5 and BL-5, the sequences are shown as SEQ ID No. 30-SEQ ID No.30 respectively). The primer synthesis was from Biometrics Ltd. Experiments prove that the primer combination 1 and the primer combination 3 have better detection capability on negative and positive samples, the combination 2 cannot work, and the combinations 4 and 5 have nonspecific amplification (figure 1). Subsequent verification shows that the primer combination 1 has poor specificity, non-specific amplification occurs in streptococcus B, streptococcus A, vibrio parahaemolyticus, haemophilus influenzae, pseudomonas aeruginosa and helicobacter pylori (figure 2), and the primer combination 3 has good specificity (figures 7 and 8). Therefore, the optimal primer combination 3 including the primers F3-3, B3-3, FIP-3, BIP-3, FL-3 and BL-3 is selected. The method comprises the following specific steps:
F3-3：GTGGGGAGCAAACAGGATT(SEQ ID No.13)
B3-3：CCTGGTAAGGTTCTTCGCG(SEQ ID No.14)
FIP-3：CGGCACTAAGCCCCGGAAAG-GTAGTCCACGCCGTAAACG(SEQ ID No 15)
BIP-3：CCTGGGGAGTACGACCGCAA-CATGCTCCACCGCTTGTG(SEQ ID No.16)
FL-3：GGCCTAACACCTAGCACTCAT(SEQ ID No.17)
BL-3：GGTTGAAACTCAAAGGAATTGACG(SEQ ID No.18)
EXAMPLE two LAMP detection reaction conditions based on 16s rRNA Streptococcus bovis primer set
The concentrations of the primers F3-3, B3-3, FIP-3, BIP-3, FL-3 and BL-3 were 0.2. Mu. Mol/L, 1.6. Mu. Mol/L, 0.8. Mu. Mol/L and 0.8. Mu. Mol/L, respectively.
The detection reagent comprises 1.4mmol/L dNTP, 20mmol/L Tris-HCl, 10mmol/L KCl and 10mmol/L (NH) 4 ) 2 SO 4 、6mmol/LMgSO 4 0.1% TritonX-100, 320U/mLBst DNA polymerase, 10. Mu. Mol/L phenol red.
In order to screen the optimal reaction temperature, the amplification temperatures were set to 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃,65 ℃, 66 ℃ and 67 ℃ respectively, and the visualization results were observed after 1h of isothermal amplification. As shown in FIG. 3, the system can produce positive amplification at 60-67 deg.C, the change can be observed by naked eyes, and the color difference between the negative control and the positive control is most obvious at 65 deg.C and 66 deg.C. At the same time, 2% agarose gel electrophoresis was used for validation, and as shown in FIG. 4, 2% agarose gel electrophoresis was used to show that the amplified band was brightest at 65 ℃ and non-specific amplification occurred at 67 ℃. Therefore, the optimal amplification temperature for LAMP detection of the 16s rRNA gene of Streptococcus bovis is 65 ℃.
In order to screen the optimal amplification time, phenol red in the reaction system is changed into SYBR Green I fluorescent dye, the fluorescence value of the reaction system is measured every 1min by using a fluorescent quantitative PCR instrument, a fluorescence curve is drawn, and the optimal reaction time is judged according to the fluorescence curve. The results are shown in FIG. 5, where the amplification reaction reached a plateau after 30min. Meanwhile, a phenol red acid-base indicator is applied to verify the color development performance of the reaction system at 65 ℃ for 10min, 20min, 30min, 40min, 50min and 60 min. As shown in FIG. 6, the visualized system developed a color after 30min of reaction. Therefore, the amplification time of LAMP detection of the streptococcus bovis 16s rRNA gene is 30-60min, and a better result can be obtained at 30min and 65 ℃.
EXAMPLE III LAMP detection based on 16s rRNA Streptococcus bovis primer set
During the LAPM reaction, dNTPs are continuously consumed by positive amplification to generate new chains and byproducts such as magnesium pyrophosphate, hydrogen ions and the like, and the pH value in the solution is reduced. Therefore, the SYBR Green I fluorescent dye in the reaction system is changed into phenol red (the color change range is pH 6.8-8.4, and the red color is changed into orange yellow) acid-base indicator, and the visual detection result is observed by naked eyes after the reaction is carried out in a water bath kettle at the temperature of 65 ℃ for 30min. As shown in fig. 7, the negative control was red and the positive control was orange-yellow. At the same time, 2% agarose gel electrophoresis was used to verify the visualization results, as shown in FIG. 8, with amplified bands visible in the positive control and no amplified bands in the negative control.
Example specificity of LAMP detection of Streptococcus bovis 16s rRNA Gene
In order to verify the specificity of LAMP detection of the 16s rRNA gene of the streptococcus bovis, 10 control strains of the streptococcus bovis, the streptococcus B, the streptococcus A, the vibrio parahaemolyticus, escherichia coli, the haemophilus influenzae, the staphylococcus aureus, the shigella, the salmonella, the pseudomonas aeruginosa, the helicobacter pylori and the like are used for carrying out experiments. Streptococcus bovis, streptococcus B and Streptococcus A are purchased from Ningbo Ming boat Biotech Co., ltd, and the rest strains are microorganisms stored in the university of Sichuan in the university of Waxi basic medicine and the institute of forensic science in the laboratory. As shown in FIG. 9, the results of the fluorescent amplification curves showed that the amplification curves were observed only in S.bovis, and no amplification curve was observed in any of the other bacteria. As shown in FIG. 10, the results of 2% agarose gel electrophoresis and fluorescence amplification curve are consistent, and only the amplified bands of Streptococcus bovis appear, but the amplified bands of other strains do not appear. Therefore, the streptococcus bovis 16srRNA gene detection established by the method has good specificity.
Example sensitive detection of Streptococcus pentasus 16s rRNA Gene
To determine the sensitivity of LAMP method for detecting the 16s rRNA gene of Streptococcus bovis, the target gene sequence of Streptococcus 16s rRNA was introduced into pUC57 plasmid (purchased from Biotechnology Ltd.) to create a standard, and the standard plasmid was diluted to 1.0X 10 8 copies/μL-1.0×10 0 Samples were graded by copies/. Mu.L for amplification detection. The results are shown in FIGS. 11, 12 and 13, using fluorescence amplification mediumThe line, the agarose gel electrophoresis and the visual detection can be carried out on 1.0 multiplied by 10 8 copies/μL-1.0×10 2 Amplification of a samples of copies/mu L shows that the method has high sensitivity for the detection of the 16s rRNA gene of the streptococcus bovis established by the 16s rRNA of the streptococcus bovis, and the detection limit is 1.0 multiplied by 10 2 copies/. Mu.L. In order to further examine the detection capability of the LAMP method, the reaction time of which the fluorescence signal intensity exceeds a certain threshold value is set as a Tq value, and the linear regression analysis is carried out on the standard gradient sample and the Tq value amplified by LAMP. As shown in FIG. 14, at 1.0X 10 8 copies/μL-1.0×10 4 Within the range of copies/mu L, the Tq value and the target gene concentration are in a linear relation, which shows that the method combines fluorescence energy to carry out quantitative detection on the streptococcus bovis.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A cattle streptococcus primer group based on 16s rRNA is characterized in that the cattle streptococcus conserved sequence 16s rRNA is utilized to design a primer group, and the primer group comprises primers F3-3, B3-3, FIP-3, BIP-3, FL-3 and BL-3; the nucleotide sequence of the F3-3 is shown as SEQ ID No. 13; the nucleotide sequence of B3-3 is shown as SEQ ID No. 14; the nucleotide sequence of FIP-3 is shown as SEQ ID No. 15; the nucleotide sequence of BIP-3 is shown in SEQ ID No. 16; the nucleotide sequence of FL-3 is shown in SEQ ID No. 17; the nucleotide sequence of BL-3 is shown in SEQ ID No. 18.
2. A LAMP detection method of Streptococcus bovis based on 16s rRNA, which is characterized in that LAMP detection is carried out on the Streptococcus bovis by using a detection reagent, amplification reaction conditions and the primer group of claim 1.
3. The LAMP detection method for Streptococcus bovis based on 16s rRNA according to claim 2, wherein the method is characterized in thatThe detection reagent comprises 1.4mmol/L dNTP Mix, 20mmol/L Tris-HCl, 10mmol/LKCl and 10mmol/L (NH) 4 ) 2 SO 4 、6mmol/LMgSO 4 0.1% Triton X-100, 320U/mLBst DNA polymerase, 10. Mu. Mol/L acid-base indicator.
4. The LAMP detection method for Streptococcus bovis based on 16s rRNA according to claim 3, wherein the acid-base indicator is selected from any one of phenol red, bromothymol blue, neutral red, phenolphthalein, cresol red, rosolic acid.
5. The LAMP detection method for Streptococcus bovis based on 16s rRNA according to claim 3, wherein the acid-base indicator is phenol red, and if the detection reagent changes from red to orange yellow, the LAMP detection method is positive for Streptococcus bovis.
6. The LAMP detection method of Streptococcus bovis based on 16s rRNA according to claim 2, wherein the concentrations of the primers F3-3, B3-3, FIP-3, BIP-3, FL-3 and BL-3 are 0.2. Mu. Mol/L, 1.6. Mu. Mol/L, 0.8. Mu. Mol/L, respectively.
7. The LAMP detection method of Streptococcus bovis based on 16s rRNA according to claim 2, characterized in that the amplification reaction time is 30-60min and the temperature is 60-67 ℃.
8. The method of 16s rRNA-based LAMP detection of Streptococcus bovis according to claim 7, wherein the optimal amplification reaction temperature is 65 ℃ and the time is 30min.
9. The LAMP detection method for Streptococcus bovis based on 16s rRNA according to claim 2, wherein the detection limit of the LAMP detection method for detecting the 16s rRNA gene of Streptococcus bovis is 1.0 x 10 2 copies/μL。
10. The use of the 16s rRNA-based Streptococcus bovis primer set according to any one of claims 1 to 9 and the LAMP detection method thereof in a Streptococcus bovis detection kit.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Non-Patent Citations (3)
| Title |
|---|
| DEGUO WANG等: "Development of Primer Sets for Loop-Mediated Isothermal Amplification that Enables Rapid and Specific Detection of Streptococcus dysgalactiae, Streptococcus uberis and Streptococcus agalactiae", INT J ENVIRON RES PUBLIC HEALTH, vol. 12, no. 6, 26 May 2015 (2015-05-26), pages 5737 * |
| 祖菲等: "牛链球菌的分离鉴定及生物信息学分析", 中国畜牧兽医, vol. 42, no. 11, 30 November 2015 (2015-11-30), pages 2903 * |
| 肖伟强等: "利用环介导恒温扩增16S rRNA对无乳链球菌快速鉴定的新方法", 实验与检验医学, vol. 36, no. 02, 15 April 2018 (2018-04-15), pages 150 - 152 * |
Cited By (1)
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|---|---|---|---|---|
| CN118086573A (en) * | 2024-04-18 | 2024-05-28 | 山东省科学院生态研究所(山东省科学院中日友好生物技术研究中心) | Universal verticillium dahliae detection kit and application thereof |
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