CN114686612A - Dual-fluorescence PCR detection primer probe group for tilapia streptococcicosis and freeze-dried kit - Google Patents

Dual-fluorescence PCR detection primer probe group for tilapia streptococcicosis and freeze-dried kit Download PDF

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CN114686612A
CN114686612A CN202210444378.5A CN202210444378A CN114686612A CN 114686612 A CN114686612 A CN 114686612A CN 202210444378 A CN202210444378 A CN 202210444378A CN 114686612 A CN114686612 A CN 114686612A
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伍建敏
罗梦萍
李中圣
罗律
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Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd
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Abstract

The invention belongs to the technical field of biological detection, discloses a dual-fluorescence PCR detection primer probe group for tilapia streptococcicosis and a freeze-drying kit, and particularly discloses a reagent, which comprises a primer group and/or a probe group. The reagent provided by the invention can be used for simultaneously detecting streptococcus agalactiae and streptococcus iniae, and can detect two pathogens of the tilapia streptococcicosis by only carrying out 1-time PCR amplification in one-tube reaction.

Description

Dual-fluorescence PCR detection primer probe set for streptococcicosis of tilapia mossambica and freeze-drying type kit
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a dual-fluorescence PCR detection primer probe set for tilapia streptococcicosis and a freeze-drying kit.
Background
Tilapia mossambica is one of the main aquaculture varieties in China, the aquaculture amount and the aquaculture scale are increased year by year, but with the increase of the aquaculture density, the incidence rate of various epidemic diseases of Tilapia mossambica also tends to increase, and the aquaculture epidemic diseases become one of the important factors for limiting the aquaculture yield. The streptococcicosis of tilapia is one of common epidemic diseases in tilapia culture, has the characteristics of high morbidity and mortality, and causes serious economic loss to tilapia culture industry. Research shows that the main pathogenic bacteria of the tilapia streptococcicosis are Streptococcus agalactiae (Streptococcus agalactiae) and Streptococcus iniae (Streptococcus iniae), and the two pathogenic bacteria usually appear alternately, so that the cultured tilapia streptococcicosis is infected by the Streptococcus. In addition, the two kinds of streptococcus can infect more than 20 kinds of marine and fresh water fishes besides tilapia, and can infect people and livestock through diseased fishes under certain conditions, and belong to pathogenic bacteria of people, livestock and fishes, so that the detection of the two kinds of tilapia streptococcus has important significance from the perspective of cultivation safety and food safety.
In the diagnosis of the tilapia streptococcicosis, the tilapia streptococcicosis is difficult to distinguish from other diseases only from external symptoms and colony forms, but conventional diagnosis methods such as bacterial culture, biochemical experiments, drug sensitive experiments, molecular detection and the like adopt plate culture as the most common detection method of the tilapia streptococcicosis at present, but the bacterial culture period usually needs 2-3 days, so that the period is long, the optimal drug application time is easily delayed, two kinds of streptococci cannot be distinguished through the colony forms, and the tilapia streptococcicosis is easily confused with other similar colonies. The currently common molecular detection methods include a common PCR method, a fluorescent PCR method, a digital PCR method, a constant temperature LAMP method, a constant temperature RAA method and the like. The detection is carried out by using the ordinary PCR in the standard of the aquatic industry in 2020, the feasibility of molecular detection is proved, but the ordinary PCR detection not only needs electrophoresis gel running, but also needs recovery sequencing to be finally judged, the period is still longer, but also the ordinary PCR is lower in detection sensitivity and specificity than a fluorescence PCR method, meanwhile, the operation steps of the ordinary PCR detection are more, a fixed detection environment is needed, the method can be basically operated by professional detection personnel only in a professional detection laboratory, and the method can only realize the purposes of quantification and can not realize the quantification, so that the clinical rapid detection and the application and popularization are not convenient. At present, only a fluorescent PCR method and a digital PCR method based on the fluorescent PCR method are available for quantitative and qualitative detection, and the digital PCR method is expensive in equipment, low in flux and not suitable for large-scale detection requirements of common detection laboratories. In the past, the problems of limited fluorescence PCR and equipment are less popularized, but in recent years, along with the application of the fluorescence PCR technology to African swine fever and new crown detection, the fluorescence PCR detection equipment is gradually made into a domestic product and a small-sized product, a large number of fluorescence PCR instruments with the advantage of cost performance or portability appear, and the basic condition for popularizing the fluorescence PCR technology to veterinary detection is met. The existing aquatic epidemic disease detection technology is still in a development stage, on one hand, many aquatic epidemic diseases lack an accurate detection method, and on the other hand, another limiting factor of accurate diagnosis and treatment of the aquatic epidemic diseases is that the conventional laboratory detection method consumes too long time, the aquatic epidemic diseases outbreak is fast, and the optimal treatment opportunity may be missed after the laboratory results are obtained.
Disclosure of Invention
The object of the first aspect of the present invention is to provide a reagent.
The object of the second aspect of the present invention is to provide a method for producing the reagent of the first aspect of the present invention.
The third aspect of the present invention is directed to a kit.
The fourth aspect of the present invention is directed to the use of the reagent of the first aspect of the present invention or the kit of the third aspect of the present invention.
The fifth aspect of the invention aims to provide a dual fluorescence PCR detection method for simultaneously detecting streptococcus agalactiae and/or streptococcus iniae.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, a reagent is provided, the reagent comprises a primer group and/or a probe group, the primer group comprises a primer 1 and a primer 2, and the probe group comprises a probe 1 and a probe 2;
wherein, the sequence of the primer 1 is as follows:
Saga-rtF: 5'-AACGGTTAATGAGGCTATTACTAG-3' (SEQ ID NO.1), or is the complement of this sequence;
Saga-rtR: 5'-AGGCTTCTACACGACTACC-3' (SEQ ID NO.2), or is the complement of this sequence;
the sequence of the primer 2 is as follows:
Sini-rtF: 5'-GGAAGTACGTTTGGAAGTCTTA-3' (SEQ ID NO.7), or is the complement of this sequence;
Sini-rtR: 5'-CGAACTAAAATCTTAGTGAAAATGA-3' (SEQ ID NO.8), or is the complement of this sequence;
the sequence of the probe 1 is as follows:
Saga-rtProbe: 5'-AGACTTCATTGCGTGCCAACCCTGAGA-3' (SEQ ID NO.3), or is the complement of this sequence;
the sequence of the probe 2 is as follows:
Sini-rtProbe: 5'-TAGGAAAGAGACGCAGTGTCAAAAGAC-3' (SEQ ID NO.9), or the complement of this sequence.
Preferably, the two ends of the probe sequence are respectively marked with a fluorescent group and a quenching group, and the fluorescent groups marked between the probe sequences are different.
Preferably, the fluorophore is at least one of FAM, HEX, HTX, VIC, TAMRA, ROX, and CY 5.
Preferably, the quencher group is at least one of BHQ1, BHQ2 and BHQ 3.
Further preferably, the fluorescent group is attached to the 5' end of the primer.
Further preferably, the quencher group is attached to the 3' end of the primer.
Further preferably, the 5 'end of the probe 1 is connected with a fluorescent group FAM, and the 3' end is connected with a quenching group BHQ 1.
Further preferably, the 5 'end of the probe 2 is connected with a fluorescent group HEX, and the 3' end is connected with a quenching group BHQ 1.
Preferably, the reagent further comprises a lyoprotectant.
Preferably, the lyoprotectant is one or more of trehalose, raffinose, mannitol, glycine and PEG.
Preferably, the freeze-drying protective agent is an aqueous solution of trehalose, raffinose, mannitol, glycine and PEG.
More preferably, the freeze-drying protective agent is an aqueous solution of 10-15% (m/v) trehalose, 1-5% (m/v) raffinose, 10-15% (m/v) mannitol, 1-4% (m/v) glycine and 5-7% (m/v) PEG.
Preferably, the reagent further comprises a PCR reaction solution.
Preferably, the PCR reaction solution includes UNG enzyme.
Further preferably, the final concentration of the UNG enzyme is 0.2-0.5U/muL.
Preferably, the PCR reaction solution further comprises calcium ions, buffer, dNTP and Taq enzyme.
Further preferably, the final concentration of the calcium ions is 1 to 3 mM.
Further preferably, dTTP in the dNTPs is replaced by dUTP.
Further preferably, the final concentration of the dNTP is 0.2-0.5 mM.
Further preferably, the final concentration of the Taq enzyme is 0.2-0.5 mM.
Preferably, the primer 1 is used for detecting streptococcus agalactiae.
Preferably, the primer 2 is used for detecting streptococcus iniae.
Preferably, the probe 1 is used for detecting streptococcus agalactiae.
Preferably, the probe 2 is used for detecting streptococcus iniae.
In a second aspect of the invention, there is provided a process for the preparation of the reagent of the first aspect of the invention, comprising the steps of: and mixing the primer and/or probe group, the freeze-drying protective agent and the PCR reaction solution, and freeze-drying to obtain the reagent.
Preferably, the freeze-drying process is pre-freezing at-50 to-35 ℃ for 2 to 4 hours, sublimating at-40 to-30 ℃ for 11 to 12 hours, and sublimating at 20 to 30 ℃ for 4 to 6 hours.
Further preferably, the freeze-drying process comprises pre-freezing at-40 to-35 ℃ for 3 to 4 hours, sublimating at-35 to-30 ℃ for 11 to 12 hours, and sublimating at 20 to 25 ℃ for 5 to 6 hours.
Compared with the traditional freeze-drying method at a fixed temperature, the sectional heating vacuum treatment of freezing-first sublimation-second sublimation can remarkably accelerate the freeze-drying process and improve the dryness of the sample; and (3) adopting a secondary sublimation method in the last two sections, drying at 20-30 ℃ to ensure that the sample is higher than the ambient temperature, ensuring that the sample cannot be pressed in the freeze dryer, and taking out the sample after freeze drying is finished without being influenced by the ambient humidity, prolonging the storage life of the freeze-dried powder and ensuring the stability of freeze drying.
In a third aspect of the invention, there is provided a kit comprising the reagents of the first aspect of the invention.
Preferably, the kit further comprises a negative control and a positive control.
Preferably, the kit further comprises a reconstitution solution; further, the double solution is ribozyme-free water.
In a fourth aspect of the present invention, there is provided a use of the reagent according to the first aspect of the present invention or the kit according to the third aspect of the present invention in any one of (1) to (6);
(1) identifying streptococcus agalactiae and/or streptococcus iniae;
(2) preparing a product for identifying streptococcus agalactiae and/or streptococcus iniae;
(3) detecting whether the sample to be detected is streptococcus agalactiae or streptococcus iniae;
(4) preparing a product for detecting whether a sample to be detected is streptococcus agalactiae and/or streptococcus iniae;
(5) detecting whether a sample to be detected is infected with streptococcus agalactiae and/or streptococcus iniae;
(6) preparing a product for detecting whether a sample to be detected is infected with streptococcus agalactiae and/or streptococcus iniae;
the above applications are useful for non-disease diagnostics.
In a fifth aspect of the present invention, there is provided a dual fluorescence PCR detection method for simultaneously detecting Streptococcus agalactiae and/or Streptococcus iniae, which is performed using the reagent of the first aspect of the present invention or the kit of the third aspect of the present invention.
Preferably, the detection method specifically comprises the following steps:
(1) extracting nucleic acid from a sample to be tested;
(2) performing a double-fluorescent PCR amplification reaction by using the nucleic acid of the step (1) as a template and using the reagent of the first aspect of the invention or the kit of the third aspect of the invention, and collecting a fluorescent signal;
(3) judging whether the sample to be detected contains streptococcus agalactiae and/or streptococcus iniae or not according to the fluorescent signal;
the above method is a non-disease diagnostic method.
Preferably, the amplification reaction procedure in step (2) is: at 45-50 ℃, for 2-4 min; 94-96 ℃ for 25-35 s; 5-10 s at 90-95 ℃, 25-35 s at 60-65 ℃ and 40-45 cycles.
Preferably, the test sample includes, but is not limited to, colonies and tissue samples containing Streptococcus agalactiae and Streptococcus iniae, such as blood, brain, spleen, liver, kidney, muscle of fish.
Preferably, the result determination method of the detection method is: on the basis of the establishment of the experiment, the sample to be detected has a typical amplification curve in an FAM channel, and the Ct value is less than or equal to 32, and the sample is judged to be streptococcus agalactiae nucleic acid positive; the sample to be detected has a typical amplification curve in the HEX channel, and the Ct value is less than or equal to 32, and the sample is judged to be streptococcus iniae nucleic acid positive; if the sample to be detected has a typical amplification curve in the FAM channel or the HEX channel and the Ct value is more than 32 and less than or equal to 38, the sample is judged to be suspicious, the sample is re-sampled or the sample is detected again after the nucleic acid amount of the sample is increased, if the sample still has the typical amplification curve in the FAM channel or the HEX channel after the re-sampling detection, the nucleic acid is judged to be positive, otherwise, the sample is negative; the tested sample has no typical amplification curve in the FAM channel or the HEX channel, and no Ct value or the Ct value is more than 38, and the tested sample is judged to be negative to the pathogenic nucleic acid of the corresponding channel.
The invention has the beneficial effects that:
the reagent provided by the invention can be used for simultaneously detecting streptococcus agalactiae and streptococcus iniae, and can detect two pathogens of the tilapia streptococcicosis by only carrying out 1-time PCR amplification in one-tube reaction.
The invention selects conserved gene sequences in two streptococcus genomes, and designs a group of primer probes respectively for detecting two tilapia streptococcus. Through Blast detection of NCBI database and verification of a large number of clinical samples, the primer probe set can specifically identify two tilapia streptococcus, the two tilapia streptococcus are not crossed, and non-specific amplification reaction with other common aquatic pathogens does not exist.
In order to identify two kinds of tilapia streptococcus simultaneously and facilitate storage and transportation in clinical use, the invention optimizes a reaction system and a reaction program, adds a freeze-drying protective agent in the middle of the reaction system, and prepares the freeze-dried type tilapia streptococcus dual-fluorescence PCR detection reagent by freeze-drying. The dual-fluorescence PCR freeze-drying detection reagent has high storage stability and convenient use, has no difference with a single reagent in the aspect of amplification efficiency, and has the detection sensitivity up to 100The copies/mu L has no cross reaction with other pathogens, and has the characteristics of high sensitivity, high specificity and high stability.
The fluorescent PCR detection reagent provided by the invention can diagnose tilapia streptococcicosis, help farmers to discover and identify the tilapia streptococcicosis earlier, facilitate timely adoption of corresponding prevention and treatment measures, reduce loss caused by the tilapia streptococcicosis, avoid drug abuse, reduce occurrence of drug-resistant strains, and play an important role in tilapia culture. In order to meet the requirement of clinical detection of aquatic products, after the fluorescent PCR detection reagent is freeze-dried, the fluorescent PCR detection reagent not only has lower storage and transportation requirements, but also is better than a liquid reagent in use convenience and stability, can be used for conventional laboratory detection, can also be used for on-site rapid detection if a rapid nucleic acid extraction reagent and rapid detection equipment are combined, generally one sample pretreatment and on-machine detection are carried out, only 1-2 hours are needed in the whole process, the detection requirements of farmers and technicians are met, and the fluorescent PCR detection reagent has wider use scenes and value in the market.
Drawings
FIG. 1 is a graph showing the amplification curve of the single fluorescent PCR reaction of Streptococcus agalactiae, 104、103、102、101、100Each represents a concentration of 104、103、102、101、100copies/. mu.L of Streptococcus agalactiae positive control, NC samples represent specific controls, negative controls and blank controls.
FIG. 2 shows a dolphinAmplification curves for the Streptococcus Single fluorescent PCR reaction, FIG. 104、103、102、101、100Each represents a concentration of 104、103、102、101、100copies/. mu.L of Streptococcus iniae positive control, NC sample represents specific control, negative control and blank control.
FIG. 3 is an amplification curve of a positive control, a specific control, a negative control and a blank control using freeze-dried dual fluorescence PCR, 10 of which4、103、102、101、100Each represents a concentration of 104、103、102、101、100copies/. mu.L of positive control, NC samples represent specific controls, negative controls and blank controls.
FIG. 4 is a graph showing the amplification curves of Streptococcus agalactiae positive control, specific control, negative control and blank control using a single fluorescent PCR reaction, 104、103、102、101、100Each represents a concentration of 104、103、102、101、100copies/. mu.L of Streptococcus agalactiae positive control, NC samples represent specific controls, negative controls and blank controls.
FIG. 5 is a graph showing the amplification curves of Streptococcus iniae positive control, specific control, negative control and blank control using single fluorescent PCR reaction 104、103、102、101、100Each represents a concentration of 104、103、102、101、100copies/. mu.L of Streptococcus iniae positive control, NC sample represents specific control, negative control and blank control.
FIG. 6 shows the freeze-dried form of dual fluorescent PCR reagent for Streptococcus tilapia.
FIG. 7 is an amplification curve of lyophilized test reagents versus positive control, specific control, negative control, and blank control at day 0.
FIG. 8 is an amplification curve of lyophilized test reagent positive control, specific control, negative control and blank control placed at 37 ℃ for 28 days.
FIG. 9 is a two-amplification electrophoresis chart of nested PCR of Streptococcus agalactiae clinical samples, wherein M represents DL2000 Marker, 1-37 represent 37 clinical samples of tilapia, and PC represents a positive control; NC stands for negative control.
FIG. 10 is a two-amplification electrophoresis chart of nested PCR of Streptococcus iniae clinical samples, wherein M represents DL2000 Marker, 1-37 represent 37 clinical samples of tilapia, and PC represents a positive control; NC stands for negative control.
Detailed Description
The present invention will now be described in detail with reference to specific examples, but the scope of the present invention is not limited thereto.
The materials, reagents and the like used in the present examples are commercially available materials and reagents unless otherwise specified.
Example 1 design and screening of primer and Probe sets
Downloading a cfb gene (CAMP factor) sequence of streptococcus agalactiae and a 16 s-23 s spacer sequence of streptococcus iniae from an NCBI gene database, comparing and sequencing by clustalw, selecting regions with high conservation, respectively designing a fluorescent PCR primer and a probe, and respectively designing a pair of common PCR primers and a pair of nested PCR primers at two sides of each primer and probe site for preparation of a positive control and confirmation of a detection result.
The designed primer and probe sequences are shown in Table 1, wherein, Saga-F1/R1 and Sini-F1/R1 are respectively common PCR primers of streptococcus agalactiae and streptococcus iniae, and Saga-F2/R2 and Sini-F2/R2 are respectively primers for nested PCR secondary amplification of streptococcus agalactiae and streptococcus iniae. The designed primer and probe sequences are compared with NCBI database, and the result shows that the similarity of the primers and probes in the table 1 and other common pathogens is lower than 80%, so that the specificity requirement of fluorescent PCR detection is met.
TABLE 1 fluorescent PCR amplification primers and Probe sequences for Streptococcus agalactiae and Streptococcus iniae
Figure BDA0003615931210000071
Figure BDA0003615931210000081
EXAMPLE 2 preparation of Positive control
Selecting a sample which is detected and sequenced according to the SCT 7235-plus 2020 Tilapia streptococcicosis diagnosis procedure and is verified to be streptococcus agalactiae or streptococcus iniae as positive, extracting sample nucleic acid by using a bacterial nucleic acid extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd., bacterial genome DNA extraction kit (DP302)), amplifying target fragments of streptococcus agalactiae and streptococcus iniae by using common PCR primers of SagaF1/Saga-R1 and Sini-F1/Sini-R1 respectively, wherein the amplification reaction system and the amplification reaction system are shown in Table 2, obtaining target fragments of 338p and 301bp respectively after electrophoresis, cutting the target fragments into gel, purifying, connecting the gel to a pMD-18T vector, transforming a DH5 alpha competent cell (DH 5 alpha, DH 528413 in Biotechnology, Cat.), picking and sequencing a colony of the positive single clone, after verification, single colony amplification culture and strain preservation are carried out, and the two strains are respectively named as DN5 alpha/pMD 18-Saga and DN5 alpha/pMD 18-Sini. When the recombinant plasmid is used as a positive control, 2 strains are respectively subjected to recovery culture, plasmids are extracted by using a bacterial plasmid extraction kit (Tiangen Biochemical technology (Beijing) Co., Ltd.), the concentration of a plasmid solution is measured by using a Thermo Scientific NanoDrop spectrophotometer, and the copy number is calculated according to the formula: copy number (copies/. mu.L) ═ 6.02X 1023(copies/mol)) × (plasmid concentration (ng/. mu.L). times.10-9) /(number of plasmid bases. times.660 (g/mol)). The two positive control samples were named pMD18-Saga and pMD18-Sini, respectively; when used as a positive control for a double assay, the concentration of 2 recombinant plasmids was adjusted to 1X 106And (3) mixing the 2 recombinant plasmids according to the ratio of 1:1 to obtain a positive control.
TABLE 2 Streptococcus iniae and Streptococcus agalactiae general PCR amplification reaction System and procedure
Figure BDA0003615931210000082
EXAMPLE 3 preparation of specific, negative and blank controls
Specific reference substance: nucleic acids of various common aquatic pathogens such as Escherichia coli (CICC23657), Salmonella (CICC10437), Vibrio parahaemolyticus (ATCC17802), Schubert's aeromonas (self-identified and preserved in the laboratory), Nocardia seriolae (self-identified and preserved in the laboratory) and Edwardsiella (self-identified and preserved in the laboratory) were extracted using a bacterial nucleic acid extraction kit (Tiangen Biochemical technology (Beijing) Ltd., bacterial genomic DNA extraction kit (DP302)), and the nucleic acids of the above pathogens were used as specificity control samples.
Negative control: the bacterial nucleic acid extraction kit is used for extracting the nucleic acid of a commercial tilapia sample which is detected to be negative according to the method of 'SCT 7235-2020 tilapia streptococcicosis diagnosis procedure', and the nucleic acid is used as a negative control.
Blank control: commercial nuclease-free water was used as a blank control.
Example 4 fluorescent PCR reaction System optimization
The 2 kinds of positive plasmids prepared in example 2 were diluted to 10 with nuclease-free water5、104、103、102、101、100copies/. mu.L, as a detection template optimized for sensitivity, and the specific control, negative control and blank control prepared in example 3 were used as detection templates optimized for specificity. The primer concentration, the probe concentration, the dNTP (dTTP is replaced by dUTP) (TAKARA, goods No. 4035) concentration, the UNG enzyme (TAKARA, goods No. 2820) concentration, the Taq enzyme (TAKARA, goods No. 9152AM) concentration, the magnesium ion concentration, the annealing extension temperature and the annealing extension time of two streptococcus single-fluorescent PCR reaction systems are respectively optimized, and the volume of the reaction system (table 3) is optimized to be 25 muL.
By single fluorescent PCR of two streptococciThe optimized single fluorescent PCR reaction system and the optimized reaction program are shown in Table 3, and the fluorescent PCR detection methods of the two streptococcus can reach 100The detection sensitivity of copies/mu L, no amplification to negative samples, blank reference substances and other common pathogenic bacteria, high detection sensitivity and specificity (figure 1 and figure 2), and effective removal of 10 percent of UNG enzyme in the system8Double dilution of the reaction product contamination.
TABLE 3 optimized Single fluorescent PCR reaction System and reaction procedure for Streptococcus agalactiae and Streptococcus iniae
Figure BDA0003615931210000091
Figure BDA0003615931210000101
Example 5 establishment and optimization of Freeze-drying Dual fluorescent PCR reaction System
The optimized single-fluorescence PCR reaction system is used as the detection performance index of the double-fluorescence PCR reaction system, and the detection sensitivity, specificity and amplification efficiency of the double-fluorescence PCR reaction system are consistent with the results of the single-fluorescence PCR reaction system. The method comprises the following specific steps:
the 2 positive plasmids prepared in example 2 (concentration: 10)6copies/. mu.L) were first treated as nuclease-free water: pMD 18-Saga: pMD18-Sini ═ 8:1:1 diluted to 105copies/. mu.L, then 105copies/. mu.L of mixed plasmid solution was diluted to 10 with a nuclease-free water gradient4、103、102、101、100copies/. mu.L as a detection template for optimizing the sensitivity of the freeze-drying dual-fluorescence PCR reaction system, and the specific reference substance, the negative reference substance and the blank reference substance prepared in example 3 as the detection template for optimizing the specificity, and the primer concentration, the probe concentration, the dNTP (dTTP-to-dUTP) concentration, the UNG enzyme concentration, the Taq enzyme concentration, the magnesium ion concentration, the freeze-drying protectant concentration, the annealing delay of the freeze-drying dual-fluorescence PCR reaction systemThe extension temperature and the annealing extension time were optimized, and the volume of the reaction system (Table 4) was optimized to 25. mu.L. Wherein the freeze-drying protective agent is an aqueous solution containing 15% (m/v) trehalose, 5% (m/v) raffinose, 10% (m/v) mannitol, 1% (m/v) glycine and 5% (m/v) PEG 2000.
After optimization, the freeze-drying double-fluorescence PCR reaction system and the reaction program are shown in table 4, the detection performance and the amplification efficiency of the optimized freeze-drying double-fluorescence PCR reaction system on a mixed sample of streptococcus agalactiae and streptococcus iniae are basically consistent with those of a single-fluorescence PCR system (table 5), and the detection sensitivity can reach 100The copies/mu L has no non-specific amplification to other common aquatic pathogenic bacteria (specific reference substances) and negative reference substances (figures 3-5), and the contained UNG enzyme can effectively remove 10 percent8The pollution of the doubly diluted reaction product can meet the detection requirement of the tilapia streptococcus.
TABLE 4 optimized lyophilized Dual fluorescence PCR reaction System and reaction procedure
Figure BDA0003615931210000102
Figure BDA0003615931210000111
Example 6A lyophilized test reagent
A lyophilized detection reagent comprising the following components: 2.5. mu.L of 10 XTaq enzyme Buffer, 2. mu.L of dNTP (containing dUTP, 2.5mM each), 1. mu.L of 25mM MgCl 21 μ L of 10 μ M Saga-rtF, 1 μ L of 10 μ M Saga-rtR, 0.5 μ L of 10 μ M Saga-rtProbe, 0.8 μ L of 10 μ M Sini-rtF, 0.8 μ L of 10 μ M Sini-rtR, 0.5 μ L of 10 μ M Sini-rtProbe, 1.5 μ L of 5U/μ L Taq enzyme, 2.5 μ L of 2U/μ L UNG enzyme, 0.9 μ L ribozyme-free water, and 5 μ L of lyoprotectant, wherein the lyoprotectant contains an aqueous solution of 15% (M/v) trehalose, 5% (M/v) raffinose, 10% (M/v) mannitol, 1% (M/v) glycine, 5% (M/v) PEG 2000.
Example 7 preparation of lyophilized detection reagents and optimization of lyophilization procedure
And (3) preparing a reaction solution of the freeze-drying detection reagent according to the embodiment 6, subpackaging the reaction solution into 200 mu L of 8-PCR tubes according to 20 mu L/tube, and freeze-drying the reaction solution in a freeze dryer to obtain the freeze-drying detection reagent. And optimizing the freeze-drying program and the freeze-drying temperature of the freeze-drying detection reagent according to the form of the freeze-drying detection reagent and the detection sensitivity and specificity of the reagent after re-melting, and selecting the optimal freeze-drying condition.
After optimization, the optimal freeze-drying procedure is pre-freezing for 3h at-40 ℃; first sublimation at-30 ℃ for 12 h; the reagent is sublimated for 5h for the second time at 25 ℃, under the freeze-drying condition, the reagent has the optimal freeze-drying shape (figure 6), after 20 mu L of nuclease-free water is added, the freeze-drying detection reagent can be re-melted quickly, and the detection sensitivity and specificity are consistent with those before freeze-drying.
EXAMPLE 8 Freeze-dried assay reagent stability test
The stability of the lyophilized detection reagent of example 6 was tested using an accelerated aging test, specifically: freeze-dried test reagent after freeze-drying, a part of the freeze-dried reagent was thawed by immediately adding 20. mu.L of nuclease-free water (day 0), and then diluted to 10 degrees after mixing with a sensitivity and specificity control (containing positive control (2 kinds of positive plasmids prepared in example 2, etc.) stored at-80 degrees C4、103、102、101、100copies/. mu.L) and NC samples (the specific control, negative control and blank control prepared in example 3)) were subjected to fluorescent PCR amplification (amplification procedure as in Table 4); and another part of the freeze-dried reagent is stored in a dark place at 37 ℃ for 28 days, 20 mu L of nuclease-free water is added into the freeze-dried detection reagent after 28 days for re-fusion, the same batch of sensitivity and specificity quality control products stored at-80 ℃ are used for carrying out fluorescence PCR amplification under the same amplification program, and the stability of the freeze-dried reagent is obtained according to the detection result.
The freeze-drying detection reagent which is aged at 37 ℃ for 28 days is detected, and as can be seen from table 5, corresponding amplification curves appear on positive reference substances with different concentrations before and after the freeze-drying detection reagent is aged at 37 ℃ and in FAM and HEX channels, Ct values have no obvious difference, and detection sensitivity can reach 100copies/. mu.L; no amplification of specific control, negative control and blank controlThe increase curves (fig. 7 and 8) show that the detection sensitivity and specificity of the lyophilized detection reagent obtained by lyophilizing the dual fluorescence PCR reaction system of example 5 are not reduced by storage, and the stability is better. The lyophilized test reagent of example 6 can be stored at 4 ℃ for more than 1 year, as calculated by the Arrhenius formula.
Table 5 lyophilized test reagent stability test
Figure BDA0003615931210000121
Note: "-" indicates no detection.
Example 9 Freeze-dried Tilapia Streptococcus agalactiae & Streptococcus iniae double fluorescence PCR detection kit
A freeze-dried dual-fluorescence PCR detection kit for tilapia streptococcus agalactiae and streptococcus iniae comprises the following components: the lyophilized detection reagent of example 6, the reconstituted solution (no ribozyme water), the positive control of example 2 and the negative control (no ribozyme water), wherein the positive control is lyophilized and diluted with no ribozyme water when used. The reaction procedure of the detection kit is shown in Table 4, and the composition condition is shown in Table 6
TABLE 6 composition of freeze-dried type Tilapia Streptococcus agalactiae and Streptococcus iniae dual fluorescence PCR detection kit
Components Dosage/reaction Amount of each box
Freeze-dried detection reagent 1T 16T/bag, 3 bags
Compound solution 20μL 1.5 mL/tube, 1 tube
Positive control 5μL 1 tube
Negative control (non-ribozyme water) 5μL 100 μ L/tube, 1 tube
EXAMPLE 10 clinical sample testing
37 tilapia samples (including tilapia with streptococcal disease clinical symptoms and tilapia without streptococcal disease clinical symptoms) are randomly selected, the freeze-dried tilapia streptococcus agalactiae and streptococcus iniae dual fluorescence PCR kit of the embodiment 9 is used for detection, nested PCR detection is simultaneously carried out by using nested PCR detection primers designed in the table 1 (common PCR detection is adopted in a standard method, the detection sensitivity is greatly different from the fluorescence PCR), the reaction system and the procedure of the nested PCR are shown in the table 7, if positive fragments appear in the nested PCR amplification, the fragments are recovered and sequenced, the sequencing is compared with an NCBI gene database, and the samples are determined to be positive when the similarity of the fragments with a reference sequence is more than 98%.
As can be seen from table 8, on the basis of the positive control and the negative control, the detection results of 37 clinical tilapia samples detected by using the freeze-dried tilapia streptococcus agalactiae and streptococcus iniae dual-fluorescence PCR kit and nested PCR of example 9 are substantially consistent (fig. 9 and 10), and no cross amplification phenomenon occurs, so that two kinds of streptococcus can be effectively distinguished; the two detection methods have slight difference, the difference result is detected to be negative on 3 samples (No. 8, No. 12 and No. 24 samples) with the Ct value higher than 38 detected by the fluorescence PCR, the 3 samples are determined to be negative after being rechecked by the kit, the low-content nucleic acid is presumed to be related to sampling randomness and other factors in the detection, and the possibility that the extremely low-content pathogen causes disease in clinic is low, so the sample with the Ct value higher than 38 detected by the kit is determined to be negative. The results prove that the freeze-dried tilapia streptococcus agalactiae and streptococcus iniae dual-fluorescence PCR kit in the example 9 has higher detection performance and reliability in clinical detection. The determination standard of the detection result of the sample by the method of the freeze-dried tilapia streptococcus agalactiae & streptococcus iniae dual fluorescence PCR kit in the embodiment 9 is as follows: on the basis of the establishment of the experiment, the sample to be detected has a typical amplification curve in an FAM channel, and the Ct value is less than or equal to 32, and the sample is judged to be streptococcus agalactiae nucleic acid positive; the sample to be detected has a typical amplification curve in the HEX channel, and the Ct value is less than or equal to 32, and the sample is judged to be streptococcus iniae nucleic acid positive; if the sample to be detected has a typical amplification curve in the FAM channel or the HEX channel and the Ct value is more than 32 and less than or equal to 38, the sample is judged to be suspicious, the sample is re-sampled or the sample is detected again after the nucleic acid amount of the sample is increased, if the sample still has the typical amplification curve in the FAM channel or the HEX channel after the re-sampling detection, the nucleic acid is judged to be positive, otherwise, the sample is negative; the tested sample has no typical amplification curve in the FAM channel or the HEX channel, and no Ct value or the Ct value is more than 38, and the tested sample is judged to be negative to the pathogenic nucleic acid of the corresponding channel.
TABLE 7 nested PCR amplification reaction System and procedure for Streptococcus iniae and Streptococcus agalactiae
Figure BDA0003615931210000131
Figure BDA0003615931210000141
TABLE 8 clinical samples fluorescence PCR detection method and nested PCR detection method detection results
Figure BDA0003615931210000142
Figure BDA0003615931210000151
Note: in the table, "+" represents positive and "-" represents negative.
Appendix: freeze-dried dual-fluorescence PCR (polymerase chain reaction) detection kit for tilapia streptococcus agalactiae and streptococcus iniae
[ USE ]
The kit adopts a double fluorescence PCR method to detect tilapia streptococcus agalactiae (FAM channel) and streptococcus iniae (HEX channel) nucleic acid (DNA) in a sample, and can be used for detecting and diagnosing tilapia streptococcus diseases and investigating epidemiology.
[ kit composition ] 48 copies/box, 25. mu.L/reaction System (detection)
Figure BDA0003615931210000152
[ PROVIDED EQUIPMENT ] FOR USE IN AN INSTALLATION
1. Reagent: a bacterial nucleic acid extraction kit adopting a column or magnetic bead method.
2. The instrument comprises the following steps: centrifuge, fluorescent PCR amplification instrument, tissue grinding instrument, refrigerator at-20 deg.C, adjustable pipettor (with range of 10 μ L, 20 μ L, 200 μ L, 1000 μ L respectively), scissors, and forceps.
3. Consumable material: suction head, disposable gloves, mask, etc.
[ MEANS FOR CARRYING OUT OF USE ]
1. When the positive control (freeze-drying) is used for the first time, firstly, the positive control is centrifuged in a centrifuge at 5000rpm for 30 seconds, then 100 mu L of the compound solution is added into a tube, the tube is tightly covered and then shaken on a shaker for 3 seconds, and the positive control is mixed, dissolved, instantaneously centrifuged and then uncovered for use. The redissolved positive control is stored at-20 ℃, is completely thawed before being used next time, and is instantaneously centrifuged for 10 seconds before being uncapped, so that the liquid is completely deposited at the bottom of the tube.
2. After the freeze-drying detection reagent is taken out, the residual reagent needs to be put back into the self-sealing bag with the drying agent in time, and the self-sealing opening is tightly sealed to prevent the reagent from being affected with damp. The unsealed lyophilized reagent package is recommended to be used up in 1, and is not recommended to be used continuously if the reagent is completely collapsed into a transparent liquid state by moisture.
3. Independent negative and positive controls are required to be arranged for each detection so as to control the quality of the reaction process and avoid false positive or false negative results.
4. In order to reduce pollution, the detection area needs to be divided, and the division into 4 operation areas is suggested: a sample preparation area, a nucleic acid extraction area, a reaction system preparation area and a PCR amplification area. All the partitions are physically isolated, tools are independently used in different areas, and gloves need to be replaced when the operation areas are replaced.
5. When adding nucleic acid templates, the suggested sequence of nucleic acid template addition is: negative control, nucleic acid to be detected and positive control; after the sample is added, each PCR reaction tube is covered tightly by caution (preventing liquid from splashing).
6. The wastes such as a sucker, gloves and the like and the detection samples used in the detection process need to be soaked in a 10% sodium hypochlorite solution (or a chlorine dioxide effervescent tablet aqueous solution) or sterilized under high pressure and then can be discarded.
7. Before the machine is installed, whether each reaction tube is tightly covered or not is checked, and the tube cover is not strictly opened after the machine is installed, so that the liquid is prevented from evaporating to form aerosol pollution; after the detection is finished, cleaning and disinfecting the working area immediately; the sample and the waste thereof are destroyed after being sterilized, and the operation thereof complies with the biological safety regulations.
8. For a special device, or when a PCR reaction procedure needs to be optimized, or a standard curve needs to be made to determine the copy number of the pathogenic nucleic acid of a sample, a technician of the company needs to be consulted.
[ sample preparation ]
In the sample preparation area:
tissue sample: taking 25-100 mg of tissue samples of fish brain, kidney, liver, spleen and the like, putting the tissue samples into a tissue grinding tube, adding steel balls for grinding and 1mL of physiological saline or PBS (if the samples are more, the volume of the physiological saline or PBS can be properly increased), grinding the tissue samples for 1-2 times on a tissue grinder, standing the tissue samples for 10min at room temperature (if the samples cannot be layered, a low-speed centrifuge can be used for proper instant centrifugation, but the rotation speed of over 3000g can not be used for centrifugation so as to avoid influencing the extraction of partial pathogenic nucleic acid), and carefully sucking 200 mu L of supernatant liquid for the subsequent nucleic acid extraction.
Pond water sample: 1.5mL of pond water sample is taken, 12000g of pond water sample is centrifuged for 3min, the supernatant is discarded, and the rest precipitate is resuspended by 200 mu L of physiological saline or PBS for subsequent nucleic acid extraction.
Separating the cultured bacteria liquid: the cultured bacteria liquid can be directly extracted according to the instruction of the current commercially available extraction kit or the common nucleic acid extraction method.
[ nucleic acid extraction ]
Performing at the nucleic acid extraction zone:
and (3) taking the sample to be detected prepared in the steps, operating according to the instruction of the nucleic acid extraction kit or the extraction steps in the common nucleic acid extraction method, and directly using the extracted sample nucleic acid for next detection or storing at minus 20 ℃ for later use.
[ SYSTEM PREPARATION ]
In the reaction system preparation area:
1. the required number of lyophilized test reagent tubes are removed or cut off for the number of test samples (including negative and positive controls), and the lyophilized reagents are then centrifuged instantaneously in an octal tube centrifuge for 10s to allow the white lyophilized reagents to settle to the bottom of the reagent tubes.
2. And opening the cover, respectively adding 20 mu L of redissolution into the freeze-dried powder, and then adding 5 mu L of each nucleic acid sample according to the sequence of the negative control, the nucleic acid of the sample to be detected and the positive control.
3. And (3) covering a pipe cover in time after the adding (one cover is suggested to be added to prevent cross contamination), uniformly mixing, and performing instant centrifugation and then loading on a machine for detection.
Note that: after the reagents are added and mixed uniformly, instantaneous centrifugation is needed to ensure that the liquid is at the bottom of the tube and has no bubbles.
[ real-time fluorescent PCR amplification ]
In the PCR amplification area:
on a fluorescent PCR instrument, selecting a FAM channel and a HEX channel, and carrying out PCR reaction according to the following reaction program:
Figure BDA0003615931210000171
[ result judgment ]
And (3) effectiveness judgment: the positive control has typical amplification curves in both FAM channel and HEX channel, and Ct value is less than or equal to 32; negative controls had no amplification curve or a Ct value of 0 in either channel. If the above conditions are simultaneously satisfied, the experiment is determined to be established, otherwise, the experiment result is invalid and needs to be detected again.
And (4) judging a result: and under the condition that the detection result is valid, judging the detection result of the sample according to the following method:
the sample to be detected has a typical amplification curve in the FAM channel, and the Ct value is less than or equal to 32, and the sample is judged to be the streptococcus agalactiae nucleic acid positive "+"; the sample to be detected has a typical amplification curve in the HEX channel, and the Ct value is less than or equal to 32, and the sample is judged to be streptococcus iniae nucleic acid positive "+".
The sample to be detected has a typical amplification curve in a FAM channel or a HEX channel, and the Ct value is more than 32 and less than or equal to 38, the sample is judged to be suspicious, and re-sampling is recommended or the nucleic acid amount of the sample is increased to 10 mu L for re-detection; a nucleic acid positive "+" is determined if it is suspected after a resampling test and there is a typical amplification curve in the FAM or HEX channel.
The tested sample has no typical amplification curve in FAM channel or HEX channel, and no Ct value or Ct value is more than 38, and the tested sample is judged as the pathogenic nucleic acid negative of the corresponding channel.
The embodiments of the present invention have been described in detail with reference to the drawings, but the present invention is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> Guangdong sea Daorhusbandry veterinary research institute Co., Ltd
<120> dual-fluorescence PCR detection primer probe group for tilapia streptococcicosis and freeze-drying type kit
<130>
<160> 13
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Claims (10)

1. A reagent, wherein the reagent comprises a primer group and/or a probe group, the primer group comprises a primer 1 and a primer 2, and the probe group comprises a probe 1 and a probe 2;
wherein, the sequence of the primer 1 is as follows:
Saga-rtF: 5'-AACGGTTAATGAGGCTATTACTAG-3', or is the complement of such a sequence;
Saga-rtR: 5'-AGGCTTCTACACGACTACC-3', or is the complement of such a sequence;
the sequence of the primer 2 is as follows:
Sini-rtF: 5'-GGAAGTACGTTTGGAAGTCTTA-3', or is the complement of such a sequence;
Sini-rtR: 5'-CGAACTAAAATCTTAGTGAAAATGA-3', or is the complement of such a sequence;
the sequence of the probe 1 is as follows:
Saga-rtProbe: 5'-AGACTTCATTGCGTGCCAACCCTGAGA-3', or is the complement of such a sequence;
the sequence of the probe 2 is as follows:
Sini-rtProbe: 5'-TAGGAAAGAGACGCAGTGTCAAAAGAC-3', or the complement of such a sequence.
2. The reagent of claim 1, further comprising a lyoprotectant; preferably, the reagent further comprises a PCR reaction solution.
3. The reagent of claim 2, wherein the lyoprotectant is one or more of trehalose, raffinose, mannitol, glycine, and PEG.
4. The reagent according to any one of claims 1 to 3, wherein the probe sequences are labeled with a fluorophore and a quencher at both ends, respectively, and the labeled fluorophores are different between the probe sequences.
5. A process for the preparation of a reagent according to any one of claims 2 to 4, comprising the steps of: and mixing the primer and/or probe group, the freeze-drying protective agent and the PCR reaction solution, and freeze-drying to obtain the reagent.
6. The preparation method of claim 5, wherein the freeze-drying procedure comprises pre-freezing at-50 to-35 ℃ for 2 to 4 hours, sublimation at-30 to-40 ℃ for 11 to 12 hours, and sublimation at 20 to 30 ℃ for 4 to 6 hours.
7. A kit comprising the reagent according to any one of claims 1 to 4; preferably, the kit further comprises a negative control and a positive control.
8. Use of the reagent according to any one of claims 1 to 4 or the kit according to claim 7 in any one of (1) to (6);
(1) identifying streptococcus agalactiae and/or streptococcus iniae;
(2) preparing a product for identifying streptococcus agalactiae and/or streptococcus iniae;
(3) detecting whether the sample to be detected is streptococcus agalactiae or streptococcus iniae;
(4) preparing a product for detecting whether a sample to be detected is streptococcus agalactiae and/or streptococcus iniae;
(5) detecting whether a sample to be detected is infected with streptococcus agalactiae and/or streptococcus iniae;
(6) preparing a product for detecting whether a sample to be detected is infected with streptococcus agalactiae and/or streptococcus iniae;
the above applications are for non-disease diagnostics.
9. A dual fluorescence PCR detection method for simultaneously detecting Streptococcus agalactiae and/or Streptococcus iniae, which is carried out by using the reagent of any one of claims 1 to 4 or the kit of claim 7.
10. The detection method according to claim 9, characterized in that it comprises in particular the steps of:
(1) extracting nucleic acid from a sample to be tested;
(2) taking the nucleic acid in the step (1) as a template, carrying out double fluorescence PCR amplification reaction by using the reagent or the kit, and collecting a fluorescence signal;
(3) judging whether the sample to be detected contains streptococcus agalactiae and/or streptococcus iniae according to the fluorescent signal;
the above method is a non-disease diagnostic method.
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