CN114686612B

CN114686612B - Primer probe group for dual fluorescence PCR detection of streptococcicosis of tilapia and freeze-drying type kit

Info

Publication number: CN114686612B
Application number: CN202210444378.5A
Authority: CN
Inventors: 伍建敏; 罗梦萍; 李中圣; 罗律
Original assignee: Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd
Current assignee: Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd
Priority date: 2022-04-26
Filing date: 2022-04-26
Publication date: 2024-05-07
Anticipated expiration: 2042-04-26
Also published as: CN114686612A

Abstract

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

Description

Primer probe group for dual fluorescence PCR detection of streptococcicosis of tilapia and freeze-drying type kit

Technical Field

The invention belongs to the technical field of biological detection, and particularly relates to a primer probe set for dual fluorescence PCR detection of streptococcicosis of tilapia and a freeze-drying kit.

Background

The tilapia is one of main aquaculture varieties in China, the culture amount and the culture scale are increased year by year, but with the increase of the culture density, the incidence of various epidemic diseases of the tilapia is also increased, and the epidemic diseases are one of important factors for limiting the culture yield. Streptococcicosis of tilapia is one of the common epidemic diseases in tilapia culture, has the characteristics of high morbidity and mortality, and causes serious economic loss for tilapia culture industry. Studies have shown that the major pathogenic bacteria of streptococcicosis in tilapia are streptococcus agalactiae (Streptococcus agalactiae) and streptococcus iniae (Streptococcus iniae), which commonly alternate, resulting in streptococcicosis in cultured tilapia. In addition, besides tilapia, the streptococcus can infect more than 20 marine and freshwater fishes, and under certain conditions, people and livestock can be infected by the diseased fishes, and pathogenic bacteria can be caused by the people, the livestock and the fishes, so that the detection of the streptococcus of the tilapia has important significance from the standpoint of cultivation safety and food safety.

In the diagnosis of streptococcicosis of tilapia, the external symptoms and colony morphology are difficult to distinguish from other diseases, while the conventional diagnosis methods such as bacterial culture, biochemical experiments, drug sensitivity experiments, molecular detection and the like are the most commonly used detection methods of streptococcicosis of tilapia at present, but the bacterial culture period usually needs 2-3 days, so that the period is long, the optimal medication time is easily delayed, two streptococcicosis cannot be distinguished through colony morphology, and the streptococcicosis is easily confused with other similar colonies. The existing 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 feasibility of molecular detection is proved by starting to detect by using common PCR in the aquatic industry standard in 2020, but the common PCR detection not only needs electrophoresis running glue, but also needs recovery sequencing to finally judge, the period is still longer, and the common PCR has lower detection sensitivity and specificity than the fluorescent PCR method, meanwhile, the common PCR detection has more operation steps and needs a fixed detection environment, and basically can only be operated by professional detection laboratory, and the method can only realize qualitative and quantitative detection, and is inconvenient for clinical rapid detection and use popularization. At present, only a fluorescent PCR method and a digital PCR method based on the fluorescent PCR method can be used for quantitative and qualitative detection, but the digital PCR method is not suitable for a large amount of detection requirements of a common detection laboratory due to expensive equipment and smaller flux. In the past, the fluorescent PCR is limited and has little popularization, but in recent years, along with the application of the fluorescent PCR technology in African swine fever and new crown detection, the fluorescent PCR detection equipment is also gradually domesticated and miniaturized, a large number of fluorescent PCR instruments with cost performance advantage or portability advantage appear, and the fluorescent PCR instrument has the basic condition of popularization of the fluorescent PCR technology to veterinary detection. At present, aquatic epidemic disease detection technology is still in a development stage, on the one hand, a plurality of aquatic epidemic diseases lack accurate detection methods, on the other hand, another limiting factor of accurate diagnosis and treatment of the aquatic epidemic diseases is that a conventional laboratory detection method takes too long time, aquatic epidemic diseases burst faster, and laboratory results possibly miss the optimal treatment opportunity.

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 preparing the reagent of the first aspect of the present invention.

The object of the third aspect of the present invention is to provide a kit.

The object of the fourth aspect of the present invention is to provide the use of a reagent according to the first aspect of the present invention or a kit according to the third aspect of the present invention.

The object of the fifth aspect of the present invention is to provide a dual fluorescence PCR detection method for simultaneously detecting Streptococcus agalactiae and/or Streptococcus iniae.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect of the present invention, there is provided a reagent comprising a primer set comprising primer 1 and primer 2 and/or a probe set comprising probe 1 and probe 2;

Wherein, the sequence of the primer 1 is as follows:

Saga-rtF:5'-AACGGTTAATGAGGCTATTACTAG-3' (SEQ ID NO. 1), or a complement of the sequence;

Saga-rtR:5'-AGGCTTCTACACGACTACC-3' (SEQ ID NO. 2), or a complement of the sequence;

The sequence of the primer 2 is as follows:

Sini-rtF:5'-GGAAGTACGTTTGGAAGTCTTA-3' (SEQ ID NO. 7), or a complement of the sequence;

Sini-rtR:5'-CGAACTAAAATCTTAGTGAAAATGA-3' (SEQ ID NO. 8), or a complement of the sequence;

The sequence of the probe 1 is as follows:

Saga-rtProbe:5'-AGACTTCATTGCGTGCCAACCCTGAGA-3' (SEQ ID NO. 3), or a complement of the sequence;

The sequence of the probe 2 is as follows:

Sini-rtProbe:5'-TAGGAAAGAGACGCAGTGTCAAAAGAC-3' (SEQ ID NO. 9), or a complement of the sequence.

Preferably, the probe sequences are labeled with a fluorescent group and a quenching group at both ends, respectively, and the labeled fluorescent groups are different from one probe sequence to another.

Preferably, the fluorophore is at least one of FAM, HEX, HTX, VIC, TAMRA, ROX and CY 5.

Preferably, the quenching 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 quenching group is attached to the 3' end of the primer.

Further preferably, the probe 1 has a fluorescent group FAM attached to the 5 'end and a quenching group BHQ1 attached to the 3' end.

Further preferably, the probe 2 has a fluorescent group HEX attached to the 5 'end and a quenching group BHQ1 attached to the 3' end.

Preferably, the agent further comprises a lyoprotectant.

Preferably, the lyoprotectant is one or more of trehalose, raffinose, mannitol, glycine and PEG.

Preferably, the lyoprotectant is an aqueous solution of trehalose, raffinose, mannitol, glycine, PEG.

Further preferably, the lyoprotectant 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, 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 UNG enzyme is 0.2 to 0.5U/. Mu.L.

Preferably, the PCR reaction solution further comprises calcium ions and buffer, dNTP, taq enzyme.

Further preferably, the final concentration of the calcium ions is 1 to 3mM.

Further preferably, dTTP in the dNTPs is replaced by dUTP.

Further preferably, the final concentration of dNTPs is 0.2 to 0.5mM.

Further preferably, the final concentration of Taq enzyme is 0.2 to 0.5mM.

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 method of preparing a reagent according to the first aspect of the invention, comprising the steps of: mixing the primer and/or probe set, the freeze-drying protective agent and the PCR reaction liquid, and freeze-drying to obtain the reagent.

Preferably, the freeze-drying procedure is pre-freezing for 2-4 h at-50 to-35 ℃, sublimating for 11-12 h at-40 to-30 ℃ and sublimating for 4-6 h at 20-30 ℃.

Further preferably, the lyophilization procedure is pre-frozen at-40 to-35 ℃ for 3-4 hours, sublimated at-35 to-30 ℃ for 11-12 hours, and sublimated at 20-25 ℃ for 5-6 hours.

Compared with the traditional fixed temperature freeze-drying method, the sectional heating vacuum treatment of freezing, primary sublimation and secondary sublimation can obviously accelerate the freeze-drying process and improve the dryness of the sample; and in the latter two stages, a secondary sublimation method is adopted, and the drying is carried out at 20-30 ℃ so that the sample is higher than the ambient temperature, the sample which cannot be capped in the freeze dryer can be ensured, the sample is not influenced by the ambient humidity in the process of taking out the sample after the freeze drying is finished, the storage period of freeze-dried powder is prolonged, and the freeze-drying stability is ensured.

In a third aspect of the invention there is provided a kit comprising the reagent 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 complex solution is water without ribozyme.

In a fourth aspect of the invention there is provided the use of a reagent according to the first aspect of the invention or a kit according to the third aspect of the 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 a 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 application is for diagnosis of non-disease.

In a fifth aspect of the invention there is provided a dual fluorescence PCR assay for simultaneous detection of Streptococcus agalactiae and/or Streptococcus iniae using the reagent of the first aspect of the invention or the kit of the third aspect of the invention.

Preferably, the detection method specifically includes the following steps:

(1) Extracting nucleic acid from a sample to be tested;

(2) Performing a double fluorescence PCR amplification reaction with the nucleic acid of step (1) as a template and using the reagent of the first aspect of the present invention or the kit of the third aspect of the present invention, and collecting fluorescent signals;

(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.

Preferably, the amplification reaction procedure in step (2) is: 45-50 ℃ for 2-4 min; 94-96 deg.c for 25-35 s; the temperature is between 90 and 95 ℃ for 5 to 10 seconds, the temperature is between 60 and 65 ℃ for 25 to 35 seconds, and the cycle is between 40 and 45.

Preferably, the samples to be tested include, but are 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 as follows: on the basis of establishment of the experiment, 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 is judged to be positive to the streptococcus agalactiae nucleic acid; 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 to be detected is judged to be positive to the streptococcus iniae nucleic acid; the sample to be detected has a typical amplification curve in the FAM channel or the HEX channel, and Ct value is smaller than or equal to 38 and is judged to be suspicious, the sample is resampled or the sample nucleic acid quantity is increased and then is detected again, if the sample is still in the FAM channel or the HEX channel after resampling and detection, the sample is judged to be positive, otherwise, the sample is judged to be negative; the sample to be detected has no typical amplification curve in FAM channel or HEX channel, and has no Ct value or Ct value > 38, and is judged to be negative for pathogenic nucleic acid in the corresponding channel.

The beneficial effects of the invention are as follows:

The reagent provided by the invention can be used for simultaneously detecting streptococcus agalactiae and streptococcus iniae, and can detect two pathogens of tilapia streptococcicosis by performing PCR amplification for 1 time in one-tube reaction.

The invention selects conserved gene sequences in the genome of two streptococcus, and designs a group of primer probes for detecting the two streptococcus tilapia respectively. Through NCBI database Blast detection and verification of a large number of clinical samples, the primer probe group can specifically identify two streptococcus tilapia, and the two streptococcus are free from cross and non-specific amplification reaction with other common aquatic pathogens.

In order to identify two kinds of streptococcus tilapia simultaneously, and to facilitate preservation 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 streptococcus tilapia dual fluorescence PCR detection reagent through 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, has detection sensitivity of 10 ⁰ 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 help farmers to find and identify the streptococcicosis of tilapia earlier, is convenient for taking corresponding prevention and treatment measures in time, reduces the loss caused by the streptococcicosis of tilapia, can avoid abuse of medicines, reduces the occurrence of drug-resistant bacterial strains, and plays an important role in tilapia culture. In order to meet the clinical detection requirement of aquatic products, after the fluorescent PCR detection reagent is freeze-dried, the requirements of preservation and transportation are lower, and the fluorescent PCR detection reagent is better than the liquid reagent in use convenience and stability, and the fluorescent PCR detection reagent not only can be used for conventional laboratory detection, but also can be used for on-site rapid detection if combined with rapid nucleic acid extraction reagent and rapid detection equipment, usually, the pretreatment of one sample is added with the detection of an upper machine, the whole process only needs 1-2 hours, and the fluorescent PCR detection reagent meets the detection requirements of a household and a technician, and has wider use scene and value in the market.

Drawings

FIG. 1 is an amplification curve of a Streptococcus agalactiae single fluorescence PCR reaction, wherein 10 ⁴、10³、10²、10¹、10⁰ represents a Streptococcus agalactiae positive control at a concentration of 10 ⁴、10³、10²、10¹、10⁰ copies/. Mu.L, and NC samples represent a specific control, a negative control and a blank control, respectively.

FIG. 2 is an amplification curve of a Streptococcus iniae single fluorescence PCR reaction, in which 10 ⁴、10³、10²、10¹、10⁰ represents a Streptococcus iniae positive control at a concentration of 10 ⁴、10³、10²、10¹、10⁰ copies/. Mu.L, and NC samples represent a specific control, a negative control and a blank control, respectively.

FIG. 3 shows amplification curves of a positive control, a specific control, a negative control and a blank control by using freeze-dried double fluorescence PCR, wherein 10 ⁴、10³、10²、10¹、10⁰ represents the positive control with the concentration of 10 ⁴、10³、10²、10¹、10⁰ copies/. Mu.L, and NC samples represent the specific control, the negative control and the blank control, respectively.

FIG. 4 shows amplification curves of Streptococcus agalactiae positive control, specific control, negative control and blank control using a single fluorescent PCR reaction, wherein 10 ⁴、10³、10²、10¹、10⁰ represents Streptococcus agalactiae positive control at a concentration of 10 ⁴、10³、10²、10¹、10⁰ copies/. Mu.L, and NC samples represent specific control, negative control and blank control, respectively.

FIG. 5 shows amplification curves of Streptococcus iniae positive control, specific control, negative control and blank control using a single fluorescent PCR reaction, wherein 10 ⁴、10³、10²、10¹、10⁰ represents Streptococcus iniae positive control at a concentration of 10 ⁴、10³、10²、10¹、10⁰ copies/. Mu.L, and NC samples represent specific control, negative control and blank control, respectively.

FIG. 6 shows the lyophilized form of the double fluorescence PCR reagent for Streptococcus tilapia.

FIG. 7 shows amplification curves of lyophilized detection reagents for positive control, specific control, negative control and blank control on day 0.

FIG. 8 shows amplification curves of lyophilized detection reagents positive control, specific control, negative control and blank control after 28 days of incubation at 37 ℃.

FIG. 9 is a double-amplification electrophoresis chart of a streptococcus agalactiae clinical sample nested PCR, wherein M represents DL2000 Marker, 1-37 represent 37 tilapia clinical samples, and PC represents a positive control; NC represents a negative control.

FIG. 10 is a double-amplification electrophoresis chart of a Streptococcus iniae clinical sample nested PCR, wherein M represents DL2000 Marker, 1-37 represent 37 tilapia clinical samples, and PC represents a positive control; NC represents a negative control.

Detailed Description

The invention will now be described in detail with reference to specific examples, without limiting the scope of the invention.

The materials, reagents and the like used in this example are commercially available materials and reagents unless otherwise specified.

Example 1 design and screening of primer and Probe set

Downloading cfb gene (CAMP factor) sequences of streptococcus agalactiae and 16 s-23 s spacer sequences of streptococcus iniae from NCBI gene database, comparing and sequencing by clustalw, selecting regions with higher conservation, respectively designing fluorescent PCR primers and probes, and respectively designing a pair of common PCR primers and a pair of nested PCR primers on two sides of each primer and probe site for preparing positive reference substances and confirming detection results.

The designed primer and probe sequences are shown in Table 1, wherein the Saga-F1/R1 and Sini-F1/R1 are common PCR primers of streptococcus agalactiae and streptococcus iniae respectively, and the Saga-F2/R2 and Sini-F2/R2 are nested PCR double-amplified primers of streptococcus agalactiae and streptococcus iniae respectively. The designed primer and probe sequences are compared by NCBI database, and the result shows that the similarity of the primer and probe in the table 1 and other various common pathogens is lower than 80%, thereby meeting the specificity requirement of fluorescent PCR detection.

TABLE 1 fluorescent PCR amplification primers and probe sequences for Streptococcus agalactiae and Streptococcus ragmitis

EXAMPLE 2 preparation of Positive control

A sample which is positive for streptococcus agalactiae or streptococcus iniae is detected and sequenced according to the SCT 7235-2020 tilapia streptococcal disease diagnosis procedure, a bacterial nucleic acid extraction kit (Tiangen biochemical technology (Beijing) limited, a bacterial genome DNA extraction kit (DP 302)) is used for extracting nucleic acid of the sample, common PCR primers SagaF1/Saga-R1 and Sini-F1/Sini-R1 are respectively used for amplifying target fragments of streptococcus agalactiae and streptococcus iniae, an amplification reaction system and an amplification reaction system are shown in table 2, 338p and 301bp target fragments are respectively obtained after electrophoresis, the target fragments are subjected to gel cutting purification and then are connected to a pMD-18T vector, DH5 alpha competent cells (biological engineering (Shanghai) stock, B528413) are converted, positive monoclonal colonies are picked up, after the amplification culture and strain preservation of single colonies are carried out after the verification, and the two strains are respectively named as DN5 alpha/D18-Saga and pM 5 alpha/Sini-DN 18 alpha. When the bacterial plasmid is used as a positive reference, 2 strains are respectively resuscitated and cultured, plasmids are extracted by using a bacterial plasmid extraction kit (Tiangen biochemical technology (Beijing) limited), the concentration of plasmid solution is measured by using a Thermo Scientific NanoDrop spectrophotometer, and the copy number is calculated according to a formula, wherein the calculation formula is as follows: copy number (copies/. Mu.L) = (6.02X10 ²³ (copies/. Mu.L))X (plasmid concentration (ng/. Mu.L). Times.10 ^-9)/(plasmid base number). Times.660 (g/mol)). Two positive control samples were designated pMD18-Saga and pMD18-Sini, respectively; when the recombinant plasmid is used as a positive reference substance for double detection, the concentration of 2 recombinant plasmids is regulated to 1X 10 ⁶ copies/. Mu.L, and then the 2 recombinant plasmids are mixed according to the ratio of 1:1 to obtain the positive reference substance.

TABLE 2 common PCR amplification reaction System and procedure for Streptococcus iniae and Streptococcus agalactiae

Example 3 preparation of specific control, negative control and blank control

Specific control: the nucleic acid of various common aquatic pathogenic bacteria such as Escherichia coli (CICC 23657), salmonella (CICC 10437), vibrio parahaemolyticus (ATCC 17802), aeromonas schubertii (laboratory self-identification preservation), nocardia quinqueen (laboratory self-identification preservation) and Edwardsiella (laboratory self-identification preservation) were extracted using a bacterial nucleic acid extraction kit (Tiangen Biotechnology (Beijing), and bacterial genome DNA extraction kit (DP 302)), and the nucleic acid of the above pathogenic bacteria was used as a specific control.

Negative control: the nucleic acid of a commercial tilapia sample, which was detected as negative according to the method of "SCT 7235-2020 streptococcicosis diagnostic procedure", was extracted using a bacterial nucleic acid extraction kit, and the nucleic acid was used as a negative control.

Blank reference: commercial nuclease-free water was used as a blank.

Example 4 fluorescent PCR reaction System optimization

The 2 positive plasmids prepared in example 2 were diluted to 10 ⁵、10⁴、10³、10²、10¹、10⁰ copies/. Mu.L respectively using no nuclease, and the specific control, negative control and blank prepared in example 3 were used as detection templates for the sensitivity optimization. The primer concentration, probe concentration, dNTP (dTTP replaced with dUTP) (TAKARA, cat. No. 4035) concentration, UNG enzyme (TAKARA, cat. No. 2820) concentration, taq enzyme (TAKARA, cat. No. 9152 AM) concentration, magnesium ion concentration, annealing extension temperature and annealing extension time of the two Streptococcus single fluorescence PCR reaction systems were optimized respectively, and the volume of the reaction system (Table 3) was optimized to 25. Mu.L.

The optimized single fluorescence PCR reaction system and reaction program are shown in Table 3, and the detection sensitivity of the two streptococcus fluorescence PCR detection methods can reach 10 ⁰ copies/. Mu.L, and the detection sensitivity and specificity (shown in figures 1 and 2) are high for negative samples, blank reference substances and other common pathogenic bacteria, and the UNG enzyme in the system can effectively remove 10 ⁸ times diluted reaction product pollution.

TABLE 3 optimized Single fluorescence PCR reaction System and reaction procedure for Streptococcus agalactiae and Streptococcus iniae

Example 5 establishment and optimization of a lyophilized Dual fluorescence PCR reaction System

And taking the optimized single fluorescent PCR reaction system as a detection performance index of a double fluorescent PCR reaction system, wherein the detection sensitivity, specificity and amplification efficiency of the double fluorescent PCR reaction system are consistent with those of the single fluorescent PCR reaction system. The method comprises the following steps:

The 2 positive plasmids prepared in example 2 (concentration of 10 ⁶ copies/. Mu.L) were prepared first according to nuclease-free water: pMD18-Saga: pMD18-Sini =8:1:1 to 10 ⁵ copies/. Mu.L, then 10 ⁵ copies/. Mu.L of the mixed plasmid solution is diluted to 10 ⁴、10³、10²、10¹、10⁰ copies/. Mu.L by a nuclease-free water gradient, and the specific control, the negative control and the blank control prepared in example 3 are used as detection templates for optimizing sensitivity of a freeze-drying dual-fluorescence PCR reaction system, and primer concentration, probe concentration, dNTP (dTTP replaced by dUTP) concentration, UNG enzyme concentration, taq enzyme concentration, magnesium ion concentration, freeze-drying protective agent concentration, annealing extension temperature and annealing extension time of the freeze-drying dual-reaction system are optimized, wherein the volume of the optimized reaction system (table 4) is 25 mu.L. Wherein the lyoprotectant is an aqueous solution containing 15% (m/v) trehalose, 5% (m/v) raffinose, 10% (m/v) mannitol, 1% (m/v) glycine, 5% (m/v) PEG 2000.

After optimization, the freeze-dried double-fluorescence PCR reaction system and the reaction program are shown in the table 4, the detection performance and the amplification efficiency of the optimized freeze-dried double-fluorescence PCR reaction system on the mixed sample of streptococcus agalactiae and streptococcus iniae are basically consistent with those of a single-fluorescence PCR system (table 5), the detection sensitivity can reach 10 ⁰ copies/mu L, the non-specific amplification is not carried out on other common aquatic pathogenic bacteria (specific reference substances) and negative reference substances (fig. 3-5), and the pollution of reaction products diluted by 10 ⁸ times can be effectively removed by the contained UNG enzyme, so that the detection requirement of the streptococcus agave can be met.

TABLE 4 optimized lyophilized Dual fluorescence PCR reaction System and reaction procedure

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 dNTPs (containing dUTP, 2.5mM),1μL25mM MgCl₂,1μL10μM Saga-rtF,1μL10μM Saga-rtR,0.5μL10μM Saga-rtProbe,0.8μL10μM Sini-rtF,0.8μL10μM Sini-rtR,0.5μL10μM Sini-rtProbe,1.5μL 5U/μL Taq enzyme each, 2.5. Mu.L of 2U/. Mu.L of UNG enzyme, 0.9. Mu.L of nuclease-free water and 5. Mu.L of lyoprotectant, wherein the lyoprotectant contains 15% (m/v) trehalose, 5% (m/v) raffinose, 10% (m/v) mannitol, 1% (m/v) glycine, 5% (m/v) PEG2000 in water.

Example 7 preparation of lyophilized detection reagent and lyophilization procedure optimization

The reaction solution of the freeze-dried detection reagent was prepared according to example 6, and the reaction solution was dispensed into 200. Mu.L/tube and placed in a PCR tube, and freeze-dried in a freeze dryer to obtain the freeze-dried detection reagent. And optimizing the freeze-drying procedure and 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 3 hours at the temperature of minus 40 ℃; sublimating for 12h at the temperature of minus 30 ℃; the second sublimation at 25 ℃ for 5 hours, under the freeze-drying condition, the reagent has the optimal freeze-drying form (figure 6), and after 20 mu L of nuclease-free water is added, the freeze-drying detection reagent can be quickly re-melted, and the detection sensitivity and specificity are consistent with those before freeze-drying.

Example 8 lyophilized detection reagent stability test

The stability of the lyophilized test reagent of example 6 was tested using an accelerated aging test, specifically: after the freeze-drying of the detection reagent, a part of the freeze-dried reagent was taken, 20. Mu.L of nuclease-free water was immediately added (day 0) for re-melting, and then fluorescence PCR amplification was performed with the sensitivity and specificity controls (containing positive controls (10 ⁴、10³、10²、10¹、10⁰ copies/. Mu.L after mixing the concentrations of 2 positive plasmids prepared in example 2) and NC samples (specificity controls, negative controls and blank controls prepared in example 3)) stored at-80 ℃ (amplification procedure is shown in Table 4); and (3) taking another part of the freeze-dried reagent, storing the freeze-dried reagent for 28 days in a dark place at 37 ℃, adding 20 mu L of nuclease-free water into the freeze-dried detection reagent after 28 days for re-melting, and carrying out fluorescent PCR amplification under the same amplification program by using the same batch of sensitivity and specificity control stored at-80 ℃, thereby obtaining the stability of the freeze-dried reagent according to the detection result.

The freeze-dried detection reagent subjected to accelerated aging at 37 ℃ for 28 days is detected, and as can be seen from table 5, the freeze-dried detection reagent has corresponding amplification curves on FAM and HEX channels for positive control substances with different concentrations before and after accelerated aging, the Ct values have no obvious difference, and the detection sensitivity can reach 10 ⁰ copies/. Mu.L; the specificity control, the negative control and the blank control have no amplification curves (figures 7 and 8), which shows that the detection sensitivity and the specificity of the freeze-dried detection reagent obtained by freeze-drying the double fluorescence PCR reaction system of the embodiment 5 can not be reduced due to storage, and the stability is better. The lyophilized test reagent of example 6 can be stored at 4℃for more than 1 year, calculated according to the Arrhenius formula.

Table 5 freeze-dried detection reagent stability test

Note that: "-" indicates undetected.

Example 9 lyophilized tilapia streptococcus agalactiae and streptococcus iniae dual fluorescence PCR detection kit

A freeze-dried tilapia streptococcus agalactiae & streptococcus iniae dual fluorescence PCR detection kit comprises the following components: the lyophilized detection reagent of example 6, the complex melt (non-ribozyme water), the positive control of example 2 and the negative control (non-ribozyme water), wherein the positive control is lyophilized, and when used, it is diluted with non-ribozyme water. The reaction procedure of the detection kit is shown in Table 4, and the composition is shown in Table 6

TABLE 6 composition of freeze-dried tilapia streptococcus agalactiae and streptococcus ragus dual fluorescence PCR detection kit

Component (A)	Dosage/reaction	Per box measurement
			Freeze-drying detection reagent	1T	16T/pack, 3 pack
Complex solution	20μL	1.5 ML/tube, 1 tube
			Positive control	5μL	1 Pipe
Negative control (non-ribozyme water)	5μL	100 Mu L/tube, 1 tube

Example 10 clinical sample detection

37 Tilapia samples (tilapia containing clinical symptoms of streptococcosis and tilapia without clinical symptoms of streptococcosis) were randomly selected, the freeze-dried tilapia streptococcus agalactiae & streptococcus iniae dual-fluorescence PCR kit of example 9 was used for detection, and simultaneously, the nested PCR detection (common PCR detection in standard method and large difference between detection sensitivity and fluorescence PCR) was performed by using nested PCR detection primers designed in Table 1, the reaction system and the procedure of nested PCR are shown in Table 7, if the nested PCR amplification shows positive fragments, the fragments were recovered and sequenced, and compared with NCBI gene database, and the similarity with reference sequence was more than 98% and confirmed as positive samples.

As can be seen from table 8, on the basis that the positive control and the negative control are established, the detection results of the freeze-dried tilapia streptococcus agalactiae & streptococcus iniae dual fluorescence PCR kit of example 9 and the nested PCR detection of 37 clinical samples of tilapia are substantially identical (fig. 9 and 10), and no cross amplification phenomenon occurs, so that the two streptococcus can be effectively distinguished; the two detection methods also have small differences, the difference results are on 3 samples (samples No. 8, no. 12 and No. 24) with Ct values higher than 38 in fluorescence PCR detection, the 3 samples are detected as negative after being checked by the kit, the nucleic acid with low content concentration is presumed to be related to factors such as randomness of sampling and the like in detection, and the possibility of clinically causing diseases with extremely low content is low, so that the samples with Ct values higher than 38 in the detection results of the kit are judged as negative. The above results prove that the freeze-dried tilapia streptococcus agalactiae and streptococcus iniae dual-fluorescence PCR kit of the embodiment 9 has higher detection performance and reliability in clinical detection. The determination criteria for the sample detection result according to the method of the freeze-dried tilapia streptococcus agalactiae & streptococcus iniae dual fluorescence PCR kit of example 9 are as follows: on the basis of establishment of the experiment, 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 is judged to be positive to the streptococcus agalactiae nucleic acid; 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 to be detected is judged to be positive to the streptococcus iniae nucleic acid; the sample to be detected has a typical amplification curve in the FAM channel or the HEX channel, and Ct value is smaller than or equal to 38 and is judged to be suspicious, the sample is resampled or the sample nucleic acid quantity is increased and then is detected again, if the sample is still in the FAM channel or the HEX channel after resampling and detection, the sample is judged to be positive, otherwise, the sample is judged to be negative; the sample to be detected has no typical amplification curve in FAM channel or HEX channel, and has no Ct value or Ct value > 38, and is judged to be negative for pathogenic nucleic acid in the corresponding channel.

TABLE 7 Streptococcus iniae and Streptococcus agalactiae nested PCR amplification reaction System and procedure

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Table 8 fluorescent PCR detection method and detection result of nested PCR detection method for clinical samples

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Note that: in the table "+" represents positive and "-" represents negative.

Appendix: freeze-dried tilapia streptococcus agalactiae and streptococcus iniae double-fluorescence PCR detection kit specification

[ Use ]

The kit adopts a double fluorescence PCR method to detect the streptococcus agalactiae (FAM channel) and streptococcus ragus (HEX channel) nucleic acid (DNA) in the sample, and can be used for detecting, diagnosing and epidemiologically investigating the streptococcicosis of the tilapia.

[ Kit composition ] 48 copies/kit, 25. Mu.L/reaction System (detection)

[ Self-contained devices ]

1. Reagent: column type or magnetic bead method bacterial nucleic acid extraction kit.

2. Instrument: centrifuge, fluorescence PCR amplification instrument, tissue grinder, -20deg.C refrigerator, adjustable pipettor (measuring range: 10 μL, 20 μL, 200 μL, 1000 μL respectively), scissors, forceps, etc.

3. Consumable: suction heads, disposable gloves, masks, and the like.

[ Use notes ]

1. When the positive control (freeze-drying) is used for the first time, the positive control (freeze-drying) is firstly centrifuged at 5000rpm for 30 seconds in a centrifuge, then 100 mu L of complex solution is added into a tube, the tube cover is closed, the shaking is carried out on an oscillator for 3 seconds, and after the complex solution is uniformly mixed and dissolved, the complex solution is instantaneously centrifuged and then the cap is opened for use. The reconstituted positive control is preserved at-20 ℃, and should be completely melted before the next use, and the reconstituted positive control is immediately centrifuged for 10 seconds before uncapping, so that the liquid is completely deposited at the bottom of the tube.

2. After the freeze-dried detection reagent is taken out, the residual reagent is required 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 wetted. The unpacked lyophilized reagent pack was recommended to run out within 1 and continued use was not recommended if the reagent completely collapsed to a transparent liquid state upon wetting.

3. Independent negative and positive controls are required to be set for each detection to control the quality of the reaction process, so that false positive or false negative results are avoided.

4. To reduce the pollution, the detection area needs to be divided, and the detection area is recommended to be divided into 4 operation areas: the sample preparation area, the nucleic acid extraction area, the reaction system preparation area and the PCR amplification area. The physical isolation is carried out among the subareas, tools are independently used in different areas, and gloves need to be replaced when the operating areas are replaced.

5. In addition of 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 addition was completed, each PCR tube was carefully (liquid was prevented from spilling) and timely capped.

6. Waste such as suction heads, gloves and the like used in the detection process and detection samples can be discarded after being soaked in 10% sodium hypochlorite solution (or chlorine dioxide effervescent tablet aqueous solution) or sterilized under high pressure.

7. Before the machine is started, checking whether each reaction tube is tightly covered or not, and after the machine is started, strictly forbidden to open the tube cover so as to prevent liquid from evaporating to form aerosol pollution; after the detection is finished, the working area is cleaned and disinfected immediately; after the sample and the waste thereof are sterilized and destroyed, the operation should strictly comply with biosafety regulations.

8. For special equipment, or when the PCR reaction program needs to be optimized, or a standard curve needs to be made to determine the copy number of the pathogenic nucleic acid of the sample, please consult the technicians of the company.

[ Sample preparation ]

In the sample preparation zone:

Tissue sample: taking 25-100 mg of tissue samples such as brain, kidney, liver and spleen of fish, 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 on a tissue grinding instrument for 1-2 times, and standing the tissue samples at room temperature for 10min (if the samples cannot be layered, a low-speed centrifuge can be used for proper instantaneous centrifugation, but a rotating speed exceeding 3000g can not be used for centrifugation so as not to affect the extraction of part of pathogenic nucleic acid), carefully sucking 200 mu L of supernatant liquid for the subsequent extraction of nucleic acid.

Pond water sample: 1.5mL of the pond water sample was taken, 12000g was centrifuged for 3min, the supernatant was discarded, and the remaining pellet was resuspended in 200. Mu.L of physiological saline or PBS for subsequent nucleic acid extraction.

Separating the cultured bacterial liquid: directly taking the cultured bacterial liquid can directly extract the nucleic acid according to the instruction of the current commercial extraction kit or the common nucleic acid extraction method.

[ Nucleic acid extraction ]

In the nucleic acid extraction region:

and (3) taking the sample to be detected prepared by the steps, and operating according to the instruction of a nucleic acid extraction kit or the extraction steps in a common nucleic acid extraction method, wherein the extracted sample nucleic acid is directly used for the next detection or is stored at minus 20 ℃ for standby.

[ System preparation ]

The preparation of the reaction system is carried out in the preparation area of the reaction system:

1. And taking out or cutting out the freeze-dried detection reagent tubes with the required quantity according to the quantity of the detection samples (containing negative and positive reference substances), and then instantly centrifuging the freeze-dried reagent in an eight-tube centrifuge for 10s to enable the white freeze-dried reagent to sink to the bottom of the reagent tube.

2. The lid was opened, 20. Mu.L of the reconstituted solution was added to the lyophilized powder, respectively, and then 5. Mu.L of each nucleic acid sample was added in the order of negative control, sample nucleic acid to be tested, positive control.

3. After the addition, a tube cover is covered in time (one cover is recommended to be added to prevent cross contamination), and the mixture is evenly mixed and is detected by a machine after instantaneous centrifugation.

Note that: after the reagent is added and mixed evenly, instantaneous centrifugation is needed to ensure that the liquid is at the bottom of the tube and has no bubbles.

[ Real-time fluorescence PCR amplification ]

In the PCR amplification region:

On a fluorescent PCR instrument, FAM channel and HEX channel were selected and PCR reactions were performed according to the following reaction procedure:

[ result determination ]

Validity judgment: the positive control has typical amplification curves in the FAM channel and the HEX channel, and the Ct value is less than or equal to 32; the negative control had no amplification curve or Ct value of 0 in all the above channels. And if the conditions are met, judging that the experiment is met, otherwise, judging that the experiment is invalid and detecting again.

And (3) result judgment: and under the condition that the detection result is effective, judging the sample detection result 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 is judged to be positive with streptococcus agalactiae nucleic acid "+"; the sample to be detected has a typical amplification curve in the HEX channel, and Ct value is less than or equal to 32, and is judged to be positive "+".

The sample to be detected has a typical amplification curve in the FAM channel or HEX channel, and Ct value is smaller than 32 and smaller than or equal to 38, and is judged to be suspicious, and re-sampling or re-sampling the sample nucleic acid amount to 10 mu L is recommended to be carried out; if the sample is suspicious and there is a typical amplification curve in either the FAM channel or HEX channel, then a positive "+" is determined.

The sample to be detected has no typical amplification curve in FAM channel or HEX channel, no Ct value or Ct value > 38, and is judged as the negative "-" of the pathogenic nucleic acid of the corresponding channel.

The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of one of ordinary skill in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

SEQUENCE LISTING

<110> Guangdong sea big animal husbandry veterinary research institute Co., ltd

<120> Double fluorescence PCR detection primer probe set for streptococcicosis of tilapia and freeze-drying type kit

<130>

<160> 13

<170> PatentIn version 3.5

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Claims

1. A reagent, characterized in that the reagent comprises a primer group and a probe group, wherein the primer group consists of a primer 1 and a primer 2, and the probe group consists of a probe 1 and a probe 2;

Wherein, the sequence of the primer 1 is as follows:

Saga-rtF:5'-AACGGTTAATGAGGCTATTACTAG-3', or a complement of the sequence;

Saga-rtR:5'-AGGCTTCTACACGACTACC-3', or a complement of the sequence;

The sequence of the primer 2 is as follows:

Sini-rtF:5'-GGAAGTACGTTTGGAAGTCTTA-3', or a complement of the sequence;

Sini-rtR:5'-CGAACTAAAATCTTAGTGAAAATGA-3', or a complement of the sequence;

The sequence of the probe 1 is as follows:

Saga-rtProbe:5'-AGACTTCATTGCGTGCCAACCCTGAGA-3', or a complement of the sequence;

The sequence of the probe 2 is as follows:

Sini-rtProbe:5'-TAGGAAAGAGACGCAGTGTCAAAAGAC-3', or a complement of the sequence;

The reagent further comprises a lyoprotectant;

The freeze-drying protective agent is an aqueous solution of 10-15% m/v trehalose, 1%m-5%m/v raffinose, 10-15% m/v mannitol, 1%m-4%m/v glycine and 5%m-7%m/vPEG;

The reagent is used for detecting streptococcus agalactiae and streptococcus iniae.

2. The reagent according to claim 1, wherein the reagent further comprises a PCR reaction solution.

3. The reagent according to claim 1 or 2, wherein the probe sequences are labeled with a fluorescent group and a quenching group at both ends, respectively, and the labeled fluorescent groups are different from probe sequence to probe sequence.

4. A method for producing the reagent according to any one of claims 1 to 3, comprising the steps of: mixing the primer and/or probe set, the freeze-drying protective agent and the PCR reaction liquid, and freeze-drying to obtain a reagent; the freeze-drying process is pre-freezing for 2-4 hours at-50 to-35 ℃, sublimating for 11-12 hours at-30 to-40 ℃ and sublimating for 4-6 hours at 20-30 ℃.

5. A kit, characterized in that it comprises the reagent according to any one of claims 1 to 3, and further comprises a negative control and a positive control.

6. Use of the reagent of any one of claims 1 to 3 or the kit of claim 5 in any one of (1) to (5);

(1) Identifying streptococcus agalactiae and streptococcus iniae;

(2) Preparing a product for identifying streptococcus agalactiae and streptococcus iniae;

(5) Preparing a product for detecting whether a sample to be detected is infected with streptococcus agalactiae and/or streptococcus iniae;

the above application is for diagnosis of non-disease.

7. A dual fluorescence PCR detection method for simultaneously detecting streptococcus agalactiae and/or streptococcus iniae, using the reagent according to any one of claims 1 to 3 or the kit according to claim 5, which is a non-disease diagnosis method.

8. The method according to claim 7, characterized in that it comprises the following steps: (1) extracting nucleic acid from a sample to be tested;

(2) Performing double fluorescence PCR amplification reaction by using the nucleic acid in the step (1) as a template and using the reagent or the kit, and collecting fluorescence signals;

the above method is a non-disease diagnostic method.