CN115747361B - Real-time fluorescence MIRA and MIRA-LFD primer group for detecting streptococcus iniae and detection method - Google Patents
Real-time fluorescence MIRA and MIRA-LFD primer group for detecting streptococcus iniae and detection method Download PDFInfo
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Abstract
The invention discloses a real-time fluorescence MIRA and MIRA-LFD primer group for detecting streptococcus iniae and a detection method, belonging to the field of molecular biology; the primer group comprises primers shown in SEQ ID NO.1 and SEQ ID NO.2 and respective probes, and the modified bases of the primers and the probes are different from the detection platform although aiming at the same target sequence. Experiments prove that the sensitivity of the two methods is 10 2 The DNA extracted from cfu/ml bacterial liquid is far lower than the incidence concentration of streptococcus iniae. Therefore, the two methods can rapidly, efficiently and sensitively detect the streptococcus iniae, and provide an effective technical means for differential diagnosis of the streptococcus iniae.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a real-time fluorescence MIRA and MIRA-LFD primer group for detecting streptococcus iniae and a detection method.
Background
Streptococcus ragmitis belongs to the class of bacillus, the order of lactobacillus, the family of streptococcus and the genus of streptococcus, is a facultative anaerobic gram-positive bacterium, is spherical or oval, has a capsule, has no flagellum, has no motility, does not form spores, and has the characteristics of wide infection host, strong infectivity, high death rate and the like. Streptococcus ragmitis can infect more than twenty kinds of fishes including freshwater fish and seawater fish, and causes great loss to the aquaculture industry. The treatment of the streptococcus iniae disease mainly depends on the use of a plurality of antibiotics, and the use of a large number of antibiotics not only leads to the drug resistance of bacteria, but also damages the stability of water bodies, so that the early prevention of the streptococcus iniae is particularly important.
The traditional bacteria identification method is mainly classified by the physiological and biochemical characteristics of bacteria, and the method has complicated steps, complex operation and time and labor consumption. Even the PCR amplification methods commonly used in recent years are required to rely on expensive laboratory instruments and skilled technicians, thereby also resulting in the inability of these methods to be applied to basic layers as well as in-situ assays.
Disclosure of Invention
The invention aims to solve the technical problem of providing a real-time fluorescence MIRA and MIRA-LFD primer group for detecting streptococcus iniae based on a multi-enzyme isothermal rapid amplification technology (MIRA) and a detection method, wherein the method can finish amplification within 15-30min based on a basic nucleic acid amplification product, and has two detection methods of a real-time fluorescence type and a test strip type. Compared with a PCR amplification method, the method greatly shortens the amplification time and improves the amplification efficiency. Compared with the agarose gel electrophoresis technology for amplification detection, the test strip type detection method does not need to rely on an expensive gel imager any more, and the detection time is also greatly shortened. Compared with the real-time fluorescence PCR method, the time required by the real-time fluorescence MIRA method is greatly shortened, and the whole time is shortened to 20-30min. Thus, rapid identification of Streptococcus iniae can be achieved, whether in the laboratory or in a relatively late-conditioned substrate.
The invention is realized by the following technical scheme:
a real-time fluorescence MIRA primer group for detecting streptococcus iniae consists of a primer 1, a primer 2 and a probe 1;
the primer 1 is as follows (a 1) or (a 2):
(a1) A single-stranded DNA molecule shown in SEQ ID NO. 1; in particular 5'-TAAAGCATTAGAAGCGGCTAAGAAAGAAG-3'
(a2) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides of SEQ ID NO.1 and has the same function as SEQ ID NO. 1;
the primer 2 is as follows (a 3) or (a 4):
(a3) A single-stranded DNA molecule shown in SEQ ID NO. 2; in particular 5'-CAATAGTTGCTTCAAGTTCTGCTTTTTCA-3'
(a4) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides of SEQ ID NO.2 and has the same function as the SEQ ID NO. 2;
the probe 1 is (a 5) or (a 6) as follows
(a5) A single-stranded DNA molecule shown in SEQ ID NO.3, specifically 5'-TTTCCAATTCAGCTTTTGTTTCTGCTAGTAGTTTCAAGGTCTT TAG-3'; and 29 th base at the 5' -end of probe 1
i6FAMdT//idSp//iBHQ1dT；
(a6) A DNA molecule with the same function as SEQ ID NO.3 through substitution and/or deletion and/or addition of one or more nucleotides of SEQ ID NO.3, and the 29 th base at the 5' end of the probe is marked with i6FAMdT// idSp// iBHQ1dT;
the invention also provides another MIRA-LFD primer group, which consists of a primer 1, a primer 2 and a probe 2;
the primer 1 is as follows (a 1) or (a 2):
(a1) A single-stranded DNA molecule shown in SEQ ID NO. 1; in particular 5'-TAAAGCATTAGAAGCGGCTAAGAAAGAAG-3'
(a2) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides of SEQ ID NO.1 and has the same function as SEQ ID NO. 1;
the primer 2 is a 5' -terminal labeled Biotin of the sequence shown in the following (a 3) or (a 4):
(a3) A single-stranded DNA molecule shown in SEQ ID NO. 2; in particular 5'-CAATAGTTGCTTCAAGTTCTGCTTTTTCA-3'
(a4) A DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides of SEQ ID NO.2 and has the same function as the SEQ ID NO. 2;
the probe 2 is a single-stranded DNA molecule shown in SEQ ID NO.3, and is marked with idSp on the 30 th base from the 5' end, 6-FAM is marked on the 5' end, and C3Spacer is marked on the 3' end.
The invention also provides a kit containing the MIRA or MIRA-LFD primer group.
The MIRA reaction system is as follows: 14.7. Mu.L of A buffer, 1. Mu.L of 10. Mu.M upstream primer, 1. Mu.L of 10. Mu.M downstream primer, 0.5. Mu.L of 10. Mu.M probe, 2. Mu.L of Streptococcus iniae DNA template, 4.55. Mu.L of ddH 2 O and 1.25. Mu.L of B buffer.
Further, the MIRA amplification reaction was performed at a temperature of 42℃for a reaction time of 20min.
The kit also comprises a nucleic acid detection test strip matched with the detection of the MIRA amplification product;
the reagent strip comprises an antibody bound to the probe labeling group or an antibody bound to the primer 2 labeling group.
It is also an object of the invention to provide a method of:
the invention provides a method for identifying or assisting in identifying whether a bacteria to be detected is streptococcus iniae, which comprises the following steps:
performing MIRA amplification on the bacteria to be detected by using the primer group, and detecting MIRA amplification products; the amplified product is then detected with a lateral flow chromatographic strip, the method being for non-diagnostic purposes.
The invention provides a method for identifying or assisting in identifying whether a sample to be tested is infected with streptococcus iniae or contains streptococcus iniae, which comprises the following steps: performing MIRA amplification on the sample to be detected by using the primer, and detecting MIRA amplification products; detecting the amplified product by using a lateral flow chromatography test strip; the method is for non-diagnostic purposes and,
in the method, the template amplified by the MIRA is nucleic acid of the bacteria to be detected or the sample to be detected.
The invention provides an amplification primer group for streptococcus iniae and a rapid detection method, which are based on a multi-enzyme isothermal rapid amplification technology (MIRA), and can be combined with lateral flow chromatography test paper to rapidly, sensitively and highly specifically detect the streptococcus iniae so as to realize macroscopic detection.
The detection sensitivity and specificity of the MIRA-LFD amplification technology of the invention are tested, and the sensitivity test result shows that the sensitivity of the kit for detecting streptococcus iniae is 10 2 cfu/ml, and has a wide detection range of at least 10 7 -10 2 Samples in the cfu/ml range can be detected. The specific detection results show that the method is used for respectively detecting the vibrio cansonii, the vibrio alginolyticus, the vibrio harveyi, the vibrio parahaemolyticus, the vibrio rotifer, the staphylococcus epidermidis, the lactococcus garvieae, the mermaid luminous bacillus, the klebsiella pneumoniae, the streptococcus agalactiae and the streptococcus ragus, and the result is only seaStreptococcus ragmitis can be well amplified, and other bacteria cannot be amplified, so that the kit has good specificity.
Compared with the prior art, the invention has the beneficial effects that:
(1) Test time can be saved: the whole test procedure of MIRA only requires 20min, which is well below 1.5 hours of qPCR and 60min of LAMP. The whole detection process of the MIRA-LFD can be completed within one hour by adding the time of sample treatment and preparation test.
(2) The reaction temperature can be reduced: MIRA only requires a constant temperature of 42℃to complete the assay, which is far below 60℃to 95℃for qPCR and 64℃for LAMP.
(3) The method is simpler and is convenient to carry: the enzyme and other necessary things needed by the amplification are freeze-dried and stored, can be placed at normal temperature for a long time, and only need to add hydrolysis buffer solution, primer, probe and template and magnesium ions to initiate reaction during the amplification, and no skilled test personnel is needed.
(4) The specificity is strong: because probes are added in the test, the specificity of the method is increased, and the LAMP method based on the fluorescent reagent has relatively poor specificity because no probes are used.
Drawings
FIG. 1 shows the basic MIRA amplification of the upstream primer and the downstream primer of the SimA gene, and the amplification results show that the effective amplification can be carried out and can be used for the amplification of subsequent experiments.
FIG. 2 is a graph showing the determination of the optimal reaction temperature and reaction time for MIRA reaction, wherein the reaction is performed at 37 ℃, 39 ℃ and 42 ℃ for real-time fluorescence MIRA amplification, and the amplification result shows that the MIRA amplification can be performed well at 3 reaction temperatures, but the initial time of MIRA amplification and the time for entering the amplification platform stage are shortest at 42 ℃, so that 42 ℃ is selected as the optimal reaction temperature for the subsequent reaction; meanwhile, when the cycle is 40 (20 min), the reaction basically enters a platform stage, so that 20min is selected as the optimal reaction time for amplification in the subsequent experiment.
FIG. 3 shows a real-time fluorescence MIRA specificity experiment, wherein DNA of 11 bacteria including Vibrio cansonii, vibrio alginolyticus, vibrio harveyi, vibrio parahaemolyticus, vibrio rotifer, staphylococcus epidermidis, lactococcus garvieae, mermaid light-emitting bacillus, klebsiella pneumoniae, streptococcus agalactiae and streptococcus ragmitis are selected as templates, and real-time fluorescence MIRA amplification detection is carried out under the optimal condition, so that only streptococcus ragmitis can be amplified to obtain a target strip, and the primer has strong specificity.
FIG. 4 is a sensitivity experiment of real-time fluorescence MIRA, selected at a concentration of 10 7 -10 0 DNA extracted from cfu/ml bacterial liquid is used as a template, and real-time fluorescence MIRA amplification is carried out at 42 ℃, and the result shows that the real-time fluorescence MIRA can detect 10 7 -10 2 cfu/ml template, where NC represents negative control.
FIG. 5 shows a specific experiment of MIRA-LFD, which selects DNA of 11 bacteria of Vibrio canescens, vibrio alginolyticus, vibrio harveyi, vibrio parahaemolyticus, vibrio rotifer, staphylococcus epidermidis, streptococcus griseus, proteus mermairei, klebsiella pneumoniae, streptococcus dysgalactiae and Streptococcus ragmitis as templates, and performs MIRA-LFD amplification detection under the best condition selected, and the result shows that only Streptococcus iniae can be amplified and detected.
FIG. 6 shows a sensitivity test of MIRA-LFD, with a concentration of 10 7 -10 1 DNA extracted from cfu/ml bacterial liquid is used as a template, MIRA-LFD amplification is carried out at 42 ℃ for 20min, and the result shows that the minimum detection limit of MIRA-LFD is 10 2 cfu/ml。
FIG. 7 shows the sensitivity of PCR amplification, in which the reaction was performed after 30cycles of amplification at 55℃and agarose gel electrophoresis, and the detection result showed that the minimum detection limit of PCR was 10 3 cfu/ml。
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions of details and forms of the technical solution of the present invention may be made without departing from the spirit and scope of the present invention, but these changes and substitutions fall within the scope of the present invention.
Example 1
MIRA primer and probe design for targeting streptococcus iniae simA gene
According to the MIRA primer design principle, primers were designed by using the simA gene sequence (GenBank: JF 330100.1) published in NCBI database as a target gene, and the designed primer sequences are as follows: the method comprises the following steps:
sim A-Fw 5'-TAAAGCATTAGAAGCGGCTAAGAAAGAAG-3', shown as SEQ ID NO. 1;
sim A-Rv 5'-CAATAGTTGCTTCAAGTTCTGCTTTTTCA-3', shown as SEQ ID NO. 2;
the amplification result is shown in FIG. 1, and the size of the target fragment is 239bp. Sequencing is the SimA sequence of streptococcus iniae.
Designing probes according to Sim A amplified sequence fragments, designing probe sequences according to real-time fluorescent MIRA probe design principle and MIRA-LFD probe design principle respectively,
real-time fluorescence MIRA probe 1 and MIRA-LFD probe 2 are respectively designed, and the specific sequences are as follows:
probe 1:
5’-TTTCCAATTCAGCTTTTGTTTCTGCTAGT/i6FAMdT//idSp//iBHQ1dT/AGTTTCAAGGTCTTTAG-3’
probe 2:
5’6-FAM-TTTCCAATTCAGCTTTTGTTTCTGCTAGTT/idSp/TAGTTTCAAGG TCTTTAG-C3 Spacer-3’
the above primers and probes were synthesized by the division of biological engineering (Shanghai).
Example 2
Establishment of real-time fluorescence MIRA detection streptococcus iniae reaction system and determination of reaction temperature and reaction time
The streptococcus iniae DNA template is obtained by extracting a nucleic acid releasing agent (Suzhou first reach gene technology Co., ltd.), is convenient for obtaining the template in subsequent experiments, reduces the time cost and reduces the dependence on instruments and equipment.
MIRA amplification was performed using a DNA isothermal rapid amplification KIT (fluorescent) (available from Fangfang Anpu future Biotechnology Co., ltd., product number WLN8203 KIT) comprising a reaction cell tube containing lyophilized enzyme powder, abuffer with base buffer (containing dNTP) and 280mM magnesium acetate B buffer.
The real-time fluorescence MIRA amplification system is specifically as follows: firstly, preparing and uniformly mixing 14.7 mu L of a basic buffer solution (A buffer), 1 mu L of each of upstream and downstream primers, 0.5 mu L of a 10 mu M probe, 2 mu L of template DNA and 4.55 mu L of sterile deionized water, adding a reaction unit tube containing freeze-dried enzyme powder, slightly uniformly mixing, dropwise adding 1.25 mu LB buffer on a tube cover, and centrifugally uniformly mixing until the total volume of a real-time fluorescence MIRA amplification reaction system is 25 mu L;
each real-time fluorescence MIRA amplification system is placed in a Bio-Rad real-time fluorescence quantitative PCR instrument to react for 50 cycles (25 min) at 37 ℃ and 39 ℃ and 42 ℃ respectively, and according to the reaction result, as shown in a graph 2, the real-time fluorescence MIRA amplification can be carried out at 3 reaction temperatures, but the initial time of MIRA amplification and the time for entering an amplification platform stage are earliest at 42 ℃, so that 42 ℃ is selected as the optimal reaction temperature for subsequent reaction; meanwhile, when the cycle is 40 (20 min), the reaction basically enters a platform stage, so that 20min is selected as the optimal reaction time for amplification in the subsequent experiment.
The optimal amplification temperature for detecting streptococcus iniae based on the real-time fluorescence MIRA technology is 42 ℃, and the reaction time is 20min (figure 2).
Example 3
Specific verification of real-time fluorescence MIRA detection of streptococcus iniae
Using the method for detecting streptococcus iniae obtained by optimizing in example 2, respectively detecting 11 marine organism pathogenic bacteria (table 1), and performing specific analysis and evaluation on the amplification primers;
TABLE 1 strains for real-time fluorescence MIRA detection specific analysis
| Strain name | Bacterial strain origin |
| Vibrio campbellii Vibrio cankeri | Preservation in this laboratory |
| Vibrio alginolyticus Vibrio alginolyticus | Preservation in this laboratory |
| Vibrio harveyi | Preservation in this laboratory |
| Vibrio Parahemolyticus Vibrio parahaemolyticus | Preservation in this laboratory |
| Vibrio rotiferianus Vibrio rotifer | Preservation in this laboratory |
| Staphylococcus epidermidis Staphylococcus epidermidis | Preservation in this laboratory |
| Staphylococcus aureus Staphylococcus aureus | Preservation in this laboratory |
| Klebsiella pneumoniae Klebsiella pneumoniae | Preservation in this laboratory |
| Lactococcus garvieae lactococcus garvieae | Preservation in this laboratory |
| Streptococcus dysgalactiae Streptococcus lactis | Preservation in this laboratory |
| Streptococcus iniae Streptococcus iniae | Preservation in this laboratory |
The specific verification result graph of the streptococcus iniae amplification primer is shown in figure 3, and the result of other 10 pathogenic bacteria is negative except that the streptococcus iniae has amplification strips, so that the amplification primer has good specificity.
Example 4
Sensitivity verification for detecting streptococcus iniae by real-time fluorescence MIRA
Inoculating Streptococcus iniae into 5mL BHI culture medium, culturing overnight at 28deg.C, counting by plate counting method, and performing 10-fold gradient dilution of pure bacterial liquid with enzyme-free sterile water to obtain Streptococcus iniae initial concentration of 10 7 CFU/mL, respectively diluted to 10 6 CFU/mL、10 5 CFU/mL、10 4 CFU/mL、10 3 CFU/mL、10 2 CFU/mL、10 1 CFU/mL. DNA extraction was performed according to the Streptococcus iniae DNA extraction method of example 2, and sensitivity detection was performed by using the Streptococcus iniae detection method optimized in example 2.
As shown in FIG. 4, the sensitivity test result shows that the minimum detection limit of the real-time fluorescence MIRA in the embodiment is 10 2 cfu/ml。
Example 5
Specificity verification for detecting streptococcus iniae by MIRA-LFD
Using the method for detecting streptococcus iniae obtained by optimizing in example 2, respectively detecting 11 marine organism pathogenic bacteria (table 1), and performing specific analysis and evaluation on the amplification primers;
the specificity verification result (figure 5) of the streptococcus iniae amplification primer shows that: besides positive amplified band results of streptococcus iniae, other 10 pathogenic bacteria are negative, which shows that the MIRA-LFD amplification primer has good specificity.
Example 6
Sensitivity verification for detecting streptococcus iniae by MIRA-LFD
The DNA which is the same as the sensitivity verification in the embodiment 2 is selected as a template for the sensitivity verification of the MIRA-LFD, the amplification reaction temperature is 42 ℃, the amplification time is 20min, and the detection is carried out by using a disposable nucleic acid test strip after the amplification is finished. The detection results are shown in FIG. 6, 10 7 -10 2 cfu/ml are positive, so the minimum detection limit of MIRA-LFD for detecting streptococcus iniae is 10 2 cfu/ml。
And meanwhile, the genomic DNA with the same concentration is used as a template for conventional PCR amplification, and the sensitivity of the two methods and the sensitivity of the PCR technology are compared.
Conventional PCR was 25. Mu.L system: 2 XTaq Master Mix (Vazyme, P111-01) 12.5. Mu.L, 1. Mu.L each of the upstream and downstream primers (10. Mu.M), 2. Mu.L of template, and the remainder were filled with sterile deionized water. PCR reaction procedure: 95 ℃ for 5min;94℃for 30s,50℃for 30s,72℃for 30s,30cycles;72℃for 5min.
The result of the sensitivity verification is shown in FIG. 7, and the minimum detection limit of conventional PCR is 10 3 cfu/ml, the results show that both methods of the invention have higher sensitivity than conventional PCR, wherein the sensitivity of real-time fluorescence MIRA and MIRA-LFD detection of Streptococcus iniae is 10 times that of conventional PCR.
Proved by verification, the amplification primer and the probe for detecting the streptococcus iniae based on the real-time fluorescence MIRA technology and the MIRA-LFD technology have the advantages of strong specificity, high sensitivity and the like; the whole amplification process does not depend on expensive experimental instruments and complex operation steps, can be detected by amplifying for 20min at the constant temperature of 42 ℃, is simple, quick and convenient, has strong specificity and high sensitivity, and has great application prospect.
While particular embodiments of the present invention have been described above, it will be understood by those skilled in the art that these are by way of example only and that various changes or modifications may be made to these embodiments without departing from the principles and spirit of the invention.
Claims (9)
1. The application of the primer probe group in preparing a kit for detecting streptococcus iniae based on a real-time fluorescence MIRA method is characterized in that the primer probe group consists of a primer 1, a primer 2 and a probe 1;
the primer 1 is as follows: 5'-TAAAGCATTAGAAGCGGCTAAGAAAGAAG-3';
the primer 2 is as follows: 5'-CAATAGTTGCTTCAAGTTCTGCTTTTTCA-3';
the probe 1 is: 5' -TTTCCAATTCAGCTTTTGTTTCTGCTAGT/i6F
AMdT/idSp/iBHQ1dT/AGTTTCAAGGTCTTTAG-3’。
2. The application of the primer probe group in preparing a kit for detecting streptococcus iniae based on a MIRA-LFD method is characterized in that the primer probe group consists of a primer 1, a primer 2 and a probe 2;
the primer is 1:5'-TAAAGCATTAGAAGCGGCTAAGAAAGAAG-3';
the primer is 2:5'-CAATAGTTGCTTCAAGTTCTGCTTTTTCA-3', the 5' -end of the primer 2 is marked with Biotin;
the probe 2 is 5' -6-FAM-TTTCCAATTCAGCTTTTGTTTCTGCTAGTT/idSp/TAGTTTC
AAGGTCTTTAG- C3 Spacer -3’。
3. The use according to claim 1, characterized in that the amplification reaction system of real-time fluorescent MIRA is as follows: 14.7. Mu.L of A buffer, 1. Mu.L of 10. Mu.M primer 1, 1. Mu.L of 10. Mu.M primer 2, 0.5. Mu.L of 10. Mu.M probe 1, 2. Mu.L of Streptococcus iniae DNA template, 4.55. Mu.L of ddH 2 O and 1.25. Mu.L of B buffer.
4. The use according to claim 2, wherein the amplification reaction system of the MIRA-LFD is as follows: 14.7. Mu.L of A buffer, 1. Mu.L of 10. Mu.M primer 2, 0.5. Mu.L of 10. Mu.M probe 2, 2. Mu.L of Streptococcus iniae DNA template, 4.55. Mu.L of ddH 2 O and 1.25. Mu.L of B buffer.
5. The use according to claim 1, wherein the amplification reaction temperature of MIRA is 42 ℃ and the reaction time is 20min.
6. The use according to claim 2, wherein the amplification reaction temperature of the MIRA-LFD is 42 ℃ and the reaction time is 20min.
7. The use of claim 2, wherein the kit further comprises a nucleic acid detection test strip for detection of MIRA amplification products in a matched manner;
the test strip comprises an antibody bound to the probe labeling group and an antibody bound to the primer 2 labeling group.
8. A method for identifying or assisting in identifying whether a test bacterium is streptococcus iniae, comprising the steps of: performing MIRA amplification on the bacteria to be detected by using the primer probe group as claimed in claim 2, and detecting MIRA amplification products; the amplified product is then detected with a lateral flow chromatographic strip, the method being for non-diagnostic purposes.
9. A method for identifying or aiding in the identification of whether a test sample is infected with or contains streptococcus iniae, the method comprising the steps of: performing MIRA amplification on a sample to be detected by using the primer probe group of claim 2, and detecting MIRA amplification products; detecting the amplified product by using a lateral flow chromatography test strip; the method is for non-diagnostic purposes, and the template for MIRA amplification is the nucleic acid of the sample to be tested.
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