CN113493850A - PCR primer probe group and kit for real-time fluorescent quantitative detection of toxoplasma gondii and eperythrozoon of pig and detection method thereof - Google Patents
PCR primer probe group and kit for real-time fluorescent quantitative detection of toxoplasma gondii and eperythrozoon of pig and detection method thereof Download PDFInfo
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Abstract
The invention discloses a PCR primer probe group for real-time fluorescent quantitative detection of porcine whole blood toxoplasma gondii and eperythrozoon, which comprises an upstream primer TG-529-F, a downstream primer TG-529-R and a toxoplasma specific fluorescent probe TG-529-P of toxoplasma gondii 529 gene, an upstream primer ES-F, a downstream primer ES-R and an eperythrozoon specific fluorescent probe ES-P of 16SrRNA of eperythrozoon. The dual fluorescent quantitative PCR kit assembled on the basis of the primer probe set can simultaneously detect the toxoplasmosis and the eperythrozoonosis of the pigs, has the advantages of high sensitivity, high specificity, stability and accuracy, can detect mixed infection at one time, and provides reliable technology and products for early diagnosis, prevention and control monitoring of clinical toxoplasmosis and eperythrozoonosis of a first-line pig farm.
Description
Technical Field
The invention relates to the technical field of PCR detection, in particular to a double PCR primer probe set and a kit for real-time fluorescent quantitative detection of toxoplasma gondii and eperythrozoon of a pig, and a detection method of the PCR kit.
Background
Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, and can invade pig body through mouth, eye, nose, respiratory tract, intestinal tract, skin, etc. The sick pig has symptoms of hyperpyrexia, lymphadenectasis, blood stasis spots or cyanosis on the head, the ears and the lower abdomen; pregnant sows suffer from abortion, stillbirth and teratogenesis. Eperythrozoonosis is a human and animal co-disease in which eperythrozoonosis is parasitized in pigs or human erythrocytes and transmitted through vectors, and can cause clinical symptoms of pig fever, anemia, jaundice, ear tip, limb and abdominal bleeding and the like. Different breeds and day-old pigs are susceptible to toxoplasma and eperythrozoon, and piglets and pregnant sows suffer the most serious harm and can die. The toxoplasmosis and the eperythrozoonosis of the pigs are frequently mixed and infected clinically, which not only reduces the quality and the yield of animal products and the fertility of animals, but also increases the treatment cost and causes serious economic loss to the pig industry in China. Therefore, in addition to enhancing the programmed immunity of the swinery, early diagnosis before onset of disease is necessary.
The diagnosis methods of the swine toxoplasmosis include a direct smear or tissue section method, an animal test method, an ELISA and the like, and the methods have more or less defects in the aspects of detection operation and detection results and cannot effectively realize early diagnosis before disease outbreak. With the development of biotechnology, diagnosis of toxoplasmosis by using fluorescence quantitative PCR and nucleic acid molecular hybridization techniques is increasingly emphasized by scientists, and the fluorescence quantitative PCR detection method can make up for the defect that an immunological method cannot be detected due to low antibody titer.
The diagnosis method of the eperythrozoonosis can be used for diagnosing by direct microscopic examination, but the false positive rate is high; the animal test method has long time consumption and high cost; serological methods are not applicable due to the large variation in antibodies to eperythrozoon; the common PCR method has the phenomena of non-specificity and poor stability in practical application; with the development of nucleic acid technology, the fluorescent quantitative PCR method has the advantages of real-time, sensitivity, safety and the like, and has high accuracy.
At present, some reports and some detection kits for detecting the two pathogens by using a fluorescence quantitative PCR method exist, but the two pathogens are detected by using a single pathogen, so that a method and a detection product for simultaneously and rapidly detecting toxoplasma gondii and eperythrozoon in pig whole blood are urgently needed to be established.
Disclosure of Invention
In order to solve the defects of the prior art, the invention designs a group of PCR primer probe sets for real-time fluorescent quantitative detection of toxoplasmosis and eperythrozoonosis of pigs, and the primer probe sets are used for simultaneously carrying out fluorescent quantitative detection on toxoplasmosis and eperythrozoonosis of pigs, so that the kit has the advantages of high sensitivity, high specificity, high accuracy and high speed, and provides reliable technology and products for early diagnosis, prevention, control and monitoring of clinical toxoplasmosis and eperythrozoonosis of a first-line pig farm.
In order to realize the aim, the PCR primer probe group for real-time fluorescence quantitative detection of the porcine whole blood toxoplasma gondii and the eperythrozoon provided by the invention comprises an upstream primer TG-529-F, a downstream primer TG-529-R and a toxoplasma specific fluorescent probe TG-529-P of the toxoplasma 529 gene, an upstream primer ES-F, a downstream primer ES-R and an eperythrozoon specific fluorescent probe ES-P of the 16SrRNA of the eperythrozoon;
an upstream primer TG-529-F: 5'-GCCACAGAAGGGACAGAAGTC-3' which is a sequence of SEQ ID NO: 1;
the downstream primer TG-529-R: 5'-GAAAAGCAGCCAAGCCGGAAA-3', which is the sequence of SEQ ID NO: 2;
the toxoplasma specific fluorescent probe TG-529-P is FAM-5'-CGCTTCCCAACCACGCCACCCTCTCAT-3' -BHQ1 which is a sequence of SEQ ID NO. 3, wherein the 5 'end is marked with FAM fluorescent reporter gene, and the 3' end is marked with BHQ1 fluorescent quenching gene;
an upstream primer ES-F: 5'-ATATGACTGAATCGTAAGATCTAGG-3', which is a sequence shown as SEQ ID NO. 4;
a downstream primer ES-R: 5'-GTGAGATAGACTTTAGGTGATTAAC-3' which is a sequence shown as SEQ ID NO. 5;
HEX-5'-CAAGTCAAGTCATCATGCCCCTTATGCC-3' -BHQ1 which is a sequence of SEQ ID NO. 6, wherein the 5 'end is marked with a HEX fluorescent reporter gene, and the 3' end is marked with a BHQ1 fluorescent quenching gene;
the invention adopts real-time fluorescence quantitative PCR technology to simultaneously detect toxoplasma gondii and eperythrozoon, establishes amplification primers and specific fluorescent probes of two pathogens as the core for ensuring the detection result, and uses Primerpermier 5.0 software to design specific primers and probes aiming at related genes of the toxoplasma gondii and the eperythrozoon according to the sequence of the toxoplasma gondii 529 and the sequence of 16SrRNA of the eperythrozoon registered in GenBank. The detection sensitivity of the primer probe group to the toxoplasmosis and the eperythrozoonosis is obviously higher than that of the conventional PCR, so that the high sensitivity, high specificity, accuracy and stability of double detection are ensured, the clinical application requirements of first-line pig farms are met, and a reliable technology is provided for early diagnosis, prevention and control monitoring of epidemic diseases.
Meanwhile, the invention also provides a PCR kit for real-time fluorescence quantitative detection of the toxoplasma gondii and the eperythrozoon of the pig, which comprises the primer probe group, the fluorescence PCR reaction solution and the positive control.
As a limitation to the above technical scheme, the positive control standard comprises a toxoplasma 529 standard and a eperythrozoon 16SrRNA standard;
the standard toxoplasma 529 is a cloning plasmid pUCm-TG of the gene segment of the toxoplasma 529, and the standard eperythrozoon 16SrRNA is a cloning plasmid pUCm-ES of the gene segment of the eperythrozoon 16 SrRNA. Extracting pathogenic RNA from the cloned plasmid of each positive control standard, performing reverse transcription to obtain cDNA, amplifying by using the primers to obtain two pathogen gene fragments, connecting the gene fragments to a pUCm-T vector by using TA cloning, and screening positive cloning after transformation to obtain the cloned plasmid with correct sequencing, wherein the final concentration of the cloned plasmids of the two standards is 1.0 multiplied by 108copies/μL。
The negative control standard is RNase-Free ddH2O。
The fast quantitative PCR reaction system of 25mL FastFire qPCR Premix (Probe) in the kit is as follows: 2 XFastfire qPCR PreMix 12.5. mu.L, toxoplasma and eperythrozoon upstream and downstream primers 0.75. mu.L each, probes 0.5. mu.L each, DNA template 3. mu.L, and RNase-Free ddH2O were added to make the total volume of the reaction system 25. mu.L.
The two pathogen gene primer probe sets designed by the invention are developed into a standardized real-time fluorescent quantitative dual PCR kit, and reliable detection products are provided for early diagnosis, prevention and control monitoring of clinical diarrhea virus diseases in first-line pig farms.
In addition, the invention also provides a detection method of the double fluorescence quantitative PCR kit for rapidly detecting the toxoplasma gondii and the eperythrozoon of the pig, which does not aim at the diagnosis and treatment of diseases, wherein the 25 muL quantitative PCR reaction system comprises: the upper and lower primers of toxoplasma gondii and eperythrozoon are 0.75 μ L each, and the final concentration is 0.3 μ M; the specific probes for the two viruses are 0.5 mu L respectively, and the final concentration is 0.2 mu M;
the PCR reaction conditions in the detection method are as follows: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 15s, collecting fluorescence signals, and finishing the reaction after 40 cycles.
As a limitation to the above technical solution, the conditions for determining that the detection result is valid are:
(1) detecting no Ct value by a negative control FAM channel and a HEX channel;
(2) the Ct value detected by a positive control FAM channel and a positive control HEX channel is less than or equal to 30.
As a limitation to the above technical solution, the determination criteria of the detection result are:
a. detecting that the Ct value is less than or equal to 40 by both FAM and HEX channels, and judging that the sample is toxoplasma gondii and eperythrozoon mixed infection;
b. the Ct value of the FAM channel is less than or equal to 40, the HEX channel has no Ct value, and the sample is judged to be toxoplasma infection positive and eperythrozoon infection negative;
c. the FAM channel has no Ct value, the HEX channel Ct value is less than or equal to 40, and the sample is judged to be toxoplasma infection negative and eperythrozoon infection positive;
d. and (3) the FAM channel and the HEX channel have no Ct value in detection, and the sample is judged to be negative to toxoplasma gondii and eperythrozoon infection.
Further limiting PCR reaction conditions and result judgment standards in the detection of the kit, and ensuring that an accurate detection result is obtained.
In conclusion, the invention develops a group of primer probe sets with high sensitivity and high specificity aiming at two pathogens of the toxoplasma gondii and the eperythrozoon according to the sequence design of the toxoplasma 529 conserved gene and the eperythrozoon 16SrRNA conserved gene, and assembles a real-time fluorescence quantitative dual PCR kit on the basis for simultaneously detecting the toxoplasma gondii and the eperythrozoon in a whole blood sample, establishes a PCR reaction system for real-time fluorescence quantitative detection of the toxoplasma gondii and the eperythrozoon in the whole blood of the pig by optimizing reaction conditions, and has important significance for simultaneously, rapidly and accurately diagnosing the two diseases.
Drawings
FIG. 1 is a 10-fold dilution gradient standard curve of a positive control standard in the dual real-time fluorescent quantitative PCR kit of the present invention;
FIG. 2-1 is a general diagram of the specificity test result of the dual real-time fluorescent quantitative PCR kit of the present invention;
FIG. 2-2 is a graph showing the results of a FAM channel (Toxoplasma gondii) specificity test in the general diagram of FIG. 2-1;
FIG. 2-3 is a graph showing the results of the HEX channel (eperythrozoon) specificity test in the general diagram of FIG. 2-1;
FIG. 3-1 is a general diagram of the stability test results of the dual real-time fluorescent quantitative PCR kit of the present invention;
FIG. 3-2 is a graph showing the results of FAM channel (Toxoplasma) stability tests in the general diagram of FIG. 3-1;
FIG. 3-3 is a graph of the results of the HEX channel (eperythrozoon) stability test in the general diagram of FIG. 3-1;
FIG. 4-1 is a general diagram of the sensitivity test results of the dual real-time fluorescent quantitative PCR kit of the present invention;
FIG. 4-2 is a graph showing the results of the FAM channel (Toxoplasma) sensitivity test in the general diagram of FIG. 4-1;
FIG. 4-3 is a graph showing the results of the sensitivity test of the HEX channel (eperythrozoon) in the general diagram of FIG. 4-1.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
The embodiment relates to design of a PCR primer probe set for real-time fluorescent quantitative detection of toxoplasma gondii and eperythrozoon of a pig, preparation of positive control clone plasmids in a dual real-time fluorescent quantitative PCR kit, kit assembly and detection application of the kit.
Primer probe set for designing toxoplasma gondii 529 gene and eperythrozoon 16SrRNA
(1) The pathogen positive gene sequences are as follows:
swine Toxoplasma 529 gene amplification sequence (177bp)
GCCACAGAAGGGACAGAAGTCGAAGGGGACTACAGACGCGATGCC GCTCCTCCAGCCGTCTTGGAGGAGAGATATCAGGACTGTAGATGGAGGC GAGGATGAGGATGAGAGGGTGGCGTGGTTGGGAAGCGACGAGAGTCG GAGAGGGAGAAGATGTTTCCGGCTTGGCTGCTTTTC, which is the sequence of SEQ ID NO. 7;
pig eperythrozoon 16SrRNA amplification sequence (147bp)
ATATGACTGAATCGTAAGATCTAGGAAGGATGGGGCCAAGTCAAGT CATCATGCCCCTTATGCCTTGGGCTGCAAACGTGCTTCAATGGTAGATTC AATGTGTGACAATCTAGCGATAGTGAGTTAATCACCTAAAGTCTATCTCA C, which is the sequence of SEQ ID NO. 8;
(2) design and synthesis of primers: specific primers and probes shown in table 1 below were designed using primerpermier 5.0 software based on the relevant gene sequences registered in GenBank.
In the invention, the length of the product corresponding to the detection primer of the toxoplasma gondii is 177bp, and the length of the product corresponding to the detection primer of the eperythrozoon is 147 bp.
Secondly, preparing a positive control standard substance in the PCR kit
And (3) removing the first strand of the genomic cDNA from the cloning plasmid of each positive control standard by a one-step method of Tiangen FastKing to synthesize a premixed kit, carrying out reverse transcription to obtain first strand cDNA, respectively amplifying by using primers to obtain two pathogen gene fragments, connecting the gene fragments to a pUCm-T vector by using TA cloning, and carrying out positive cloning screening after transformation to obtain the cloning plasmid with correct sequencing. In the process of screening positive clones, a clone plasmid with correct sequencing is named as pUCm-XXX, and the positive plasmid is used as a standard substance for real-time fluorescent quantitative PCR amplification.
The first step is as follows: primer synthesis
The primer sequences are shown in Table 1 and synthesized by Biotechnology engineering (Shanghai) GmbH.
The second step is that: total RNA extraction
And (3) mixing 200 mu L of known positive samples containing toxoplasma gondii and eperythrozoon respectively, placing the mixture into a 1.5mL centrifuge tube, extracting by using a high-efficiency blood total RNA extraction kit of Tiangen company to obtain blood total RNA, quickly carrying out electrophoresis to detect the RNA integrity, and storing the blood total RNA at-80 ℃ for later use.
The third step: first Strand cDNA Synthesis
First strand cDNA was obtained by reverse transcription using a premixed kit for first strand synthesis of genomic cDNA using the FastKing one-step method from Tiangen corporation according to the instructions.
The fourth step: cloning by PCR
According to the operation instruction of 2 XTaq PCR MasterMix II kit of Tiangen company, the first strand cDNA is used as a template, the primers are respectively amplified to obtain two pathogen gene fragments, the gene fragments are connected to a pUCm-T vector by TA cloning, and positive cloning is screened after transformation to obtain a cloning plasmid with correct sequencing.
The fifth step: positive control standard
Quantifying the mass concentration of positive control with correct sequencing by Biotechnology engineering (Shanghai) GmbH with Nanodrop2000 according to formula 6.02 × 1023×(X ng/μL×10-9) /(DNA length. times.660) converted to copy number, the plasmids for toxoplasma and eperythrozoon positive clones were 7X 10, respectively11Copies/. mu.L and 6X 1011Copies/. mu.L, with RNase-Free ddH2O dilution of the recombinant plasmids to 2.0X 108Copies/uL, then mixing the two positive plasmids according to the proportion of 1:1 to prepare a positive control standard substance, so that the final concentration of the positive plasmids of the toxoplasma gondii and the eperythrozoon positive plasmids in the mixture is 1.0 multiplied by 108Copies/uL。
Three, assemble kit
The double fluorescence quantitative PCR kit comprises the primers, the probe and the 2 xFastFire qPCR Premix in the technical scheme, wherein the water is RNase-Free ddH2And O. In the present invention, the primer and probe sequences (see Table 1) were synthesized by Biotechnology (Shanghai) Ltd. The 2 xFastFire qPCR Premix is preferably a FastFire qPCR Premix kit (probe method) purchased from Tiangen corporation, the premixed solution contains hot start enzyme and a unique PCR buffer solution, the DNA can be rapidly and sensitively detected by fluorescent quantitative PCR by using a sequence specific probe, and the running time can be shortened by more than 60 percent by using the product to carry out double fluorescent quantitative PCR reaction.
The fast quantitative PCR reaction system of FastFire is as follows: 2 XFastfire qPCR PreMix 12.5. mu.L, toxoplasma and eperythrozoon upstream and downstream primers 0.75. mu.L, probes 0.5. mu.L, DNA template 3. mu.L, and RNase-Free ddH2O to a total volume of 25. mu.L. The toxoplasma gondii, the eperythrozoon upstream primer and the eperythrozoon downstream primer are used in the kit at the final concentration of 0.3 mu M, and each specific fluorescent probe is used in the kit at the final concentration of 0.2 mu M. RNase-Free ddH was used as a negative control2O。
The PCR reaction conditions of the kit are as follows: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 15s, collecting fluorescence signals, circulating denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 15s, collecting fluorescence signals, and finishing the reaction after 40 cycles; the reaction program can make the specific primers of two pathogens efficiently amplify the target segment in a short time, save the detection time and ensure the specific combination of the primers and the template.
The conditions for judging the detection result to be effective by using the kit need to be as follows: simultaneously, the detection of negative control FAM channel and HEX channel without Ct value is satisfied (1); (2) the Ct value detected by a positive control FAM channel and a positive control HEX channel is less than or equal to 30.
Fourth, the amplification efficiency of the kit is verified
Toxoplasmosis and eperythrozoon positive control standards were diluted 10-fold in gradient: 108～101Copies/. mu.L, each gradient was set to 2 replicates, double fluorescent quantitative PCR was performed, and a standard curve was prepared based on the amplification results.
The amplification efficiency verification result shows that: the gradient amplification standard curves (FAM and HEX channels) diluted by 10 times of the positive control standard substance are shown in figure 1 of the accompanying drawing, standard curves of the toxoplasma gondii and the eperythrozoon are marked as a and b respectively, and parameter data of each standard curve are shown in the following table 2.
Fifth, specificity test for detecting toxoplasma gondii and eperythrozoon
In order to detect the specificity of the kit, the kit provided by the invention is used for detecting 6 pathogens such as porcine isospora, mycoplasma synovium, cryptosporidium parvum, babesia microti, porcine circovirus type II, porcine reproductive and respiratory syndrome virus and the like.
The result of the specificity test shows that: the kit only amplifies toxoplasma gondii and eperythrozoon, which shows that the kit can specifically amplify without cross reaction with porcine isospora, mycoplasma synovium, cryptosporidium parvum, babesia microti, porcine circovirus type II and porcine reproductive and respiratory syndrome virus, and the detection result is shown in the attached drawing of figures 2-1-2-3, wherein figure 2-1 is a general diagram of the specific test result, amplification curves of the toxoplasma gondii and the eperythrozoon are marked as a and b respectively, and 6 pathogens such as the porcine isospora, the synovial mycoplasma, the cryptosporidium parvum, the babesia microti, the porcine circovirus type II and the porcine reproductive and respiratory syndrome virus are not amplified and are basically in a horizontal line; FIG. 2-2 is a graph showing the results of FAM channel (Toxoplasma gondii) specificity test, and FIGS. 2-3 are graphs showing the results of HEX channel (Eperythrozoon) specificity test; therefore, the dual fluorescent quantitative PCR reaction system constructed in the invention has better specificity.
Sixthly, stability test for detecting toxoplasma and eperythrozoon
Selecting positive control standard substance of toxoplasma gondii and eperythrozoon with three concentrations of 108Copies/μL、107 Copies/μL、106Copies/. mu.L, run for each run, and run-to-run replicates. The batch repeat test is to repeat the positive control standard substance in the same double-fluorescence quantitative PCR for 2 times; the batch-to-batch repeat test was a double fluorescent quantitative PCR test performed in 2 different time trials. The coefficient of variation (CV (standard deviation/average value) of different nucleic acid concentrations of the positive control standard sample is less than 4%, which shows that the detection process of the invention has better stability. The detection results are shown in table 3 and attached figures 3-1 to 3-3, wherein figure 3-1 is a general stability test result diagram, figure 3-2 is a stability test result diagram of FAM channel (toxoplasma gondii), and figure 3-3 is a stability test result diagram of HEX channel (eperythrozoon).
Seventh, test for detecting Toxoplasma and Eperythrozoon sensibility
Plasmid standards for positive clones of Toxoplasma gondii and Eperythrozoon in 10-fold serial dilutions (1.0X 10)8～1.0×101Copies/. mu.L) as template, 2 replicates per dilution were set up, and double fluorescent quantitative PCR detection sensitivity was performed.
Test results show that the detection method can detect 1.0 multiplied by 108～1.0×101Copy amount of Copies/. mu.L of Copies of Copies/μ L is shown in FIGS. 4-1 to 4-3, wherein FIG. 4-1 is a general diagram of sensitivity test results, and FIG. 4-2 is a FAM channel (Bow)Figure) sensitivity test result chart, fig. 4-3 is a graph of the HEX channel (eperythrozoon) sensitivity test result chart, and the detection range of each curve in fig. 4-1, 4-2 and 4-3 from left to right corresponds to 1.0 × 108～1.0×101Copies/. mu.L were decreased 10-fold in sequence. The sensitivity of the method can detect samples with the lowest 30 copy number nucleic acid content.
Eight, double real-time fluorescent quantitative PCR detection example for toxoplasma and eperythrozoon
The first step is as follows: primer synthesis
The primer sequences are shown in Table 1 and synthesized by Biotechnology engineering (Shanghai) GmbH.
The second step is that: genomic DNA extraction
Each 200. mu.L of a known positive sample (pig whole blood) containing Toxoplasma gondii and Eperythrozoon was mixed and placed in a 1.5mL centrifuge tube, and whole blood DNA was extracted using a blood genome DNA extraction kit from Tiangen Biochemical technology (Beijing) Co., Ltd according to the product instructions.
The third step: dual fluorescent quantitative PCR amplification
The double fluorescent quantitative PCR reaction system and the reaction program are operated with the assembly kit.
The fourth step: determination of detection result
a. And (3) determining that the sample is toxoplasma gondii and eperythrozoon mixed infection if the Ct value detected by both FAM and HEX channels is less than or equal to 40.
b. And if the Ct value of the FAM channel is less than or equal to 40 and the HEX channel has no Ct value, the sample is judged to be toxoplasma infection positive and eperythrozoon infection negative.
c. And if the FAM channel has no Ct value and the HEX channel has a Ct value of less than or equal to 40, determining that the sample is toxoplasma gondii infection negative and eperythrozoon infection positive.
d. And if the FAM channel and the HEX channel do not have Ct values, the sample is judged to be negative to toxoplasma gondii or eperythrozoon infection.
Test for detecting toxoplasmosis and eperythrozoon in clinical samples
The first step is as follows: primer synthesis
The primer sequences are shown in Table 1 and synthesized by Biotechnology engineering (Shanghai) GmbH.
The second step is that: sample collection
75 parts of whole blood samples of the pig farm in Jingjin Ji area are collected.
The third step: genomic DNA extraction
200. mu.L of each of known positive samples (pig whole blood) containing Toxoplasma gondii and eperythrozoon was mixed and placed in a 1.5mL centrifuge tube, and genomic DNA of a whole blood sample was extracted using a blood genomic DNA extraction kit from Tiangen Biochemical technology (Beijing) Co., Ltd according to the product instructions.
The fourth step: general PCR detection and double fluorescent quantitative PCR
The general PCR assay utilizes the Tiangen 2 × Taq Plus PCR Mix kit. The common PCR reaction system is as follows: 2 XTaq Plus PCR Mix 12.5. mu.L, 10. mu.M upstream and downstream primers (common PCR upstream and downstream primers as well as fluorescent upstream and downstream primers) 1. mu.L each, 3. mu.L DNA template, RNase-Free ddH2O was added to the total volume of the reaction system of 25. mu.L, and PCR amplification was carried out separately. The reaction is carried out in a PCR amplification instrument, and the reaction parameters are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s, 30 cycles, and re-extension at 72 ℃ for 5 min. mu.L of the amplification product was subjected to 1.5% agarose electrophoresis.
The double fluorescent quantitative PCR process is the same as the above-mentioned kit assembly operation.
The detection results of 75 samples to be detected are shown in Table 4 below:
the detection result of the single real-time fluorescent quantitative PCR method with the best detection precision is regarded as a standard, so that the number of the positive samples detected by the kit is completely consistent with the detection result of the single real-time fluorescent quantitative PCR method, and the mixed detection result detected by the kit is completely consistent with the detection result of the single real-time fluorescent quantitative PCR method.
From the above results it can be seen that: the detection effect of the invention is obviously better than that of the common PCR detection, the occurrence of false negative can be reduced, meanwhile, the mixed infection can be detected by one experiment, the secondary PCR verification is reduced, the subsequent treatment of PCR amplification is not needed, the time is greatly saved, and the working efficiency is improved.
In conclusion, the gene primer probe group has high specificity and high sensitivity for simultaneously detecting target fragments of two pathogens, i.e. toxoplasma and eperythrozoon in the same PCR reaction system, has a detection effect obviously better than that of common PCR, and provides reliable technology and products for early diagnosis, prevention, control and monitoring of clinical toxoplasmosis and eperythrozoonosis in first-line pig farms.
Sequence listing
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Claims (6)
1. A group of PCR primer probe group for real-time fluorescent quantitative detection of toxoplasmosis and eperythrozoon of pig is characterized in that: comprises an upstream primer TG-529-F, a downstream primer TG-529-R and a toxoplasma specific fluorescent probe TG-529-P of the toxoplasma 529 gene, an upstream primer ES-F, a downstream primer ES-R and a eperythrozoon specific fluorescent probe ES-P of the 16SrRNA of the eperythrozoon;
the sequence of the TG-529-F is shown as SEQ ID NO 1;
the sequence of the TG-529-R is shown as SEQ ID NO. 2;
the sequence of the TG-529-P is shown as SEQ ID NO. 3, wherein the 5 'end is marked with FAM fluorescent reporter gene, and the 3' end is marked with BHQ1 fluorescent quenching gene;
the sequence of the ES-F is shown as SEQ ID NO. 4;
the sequence of the ES-R is shown as SEQ ID NO. 5;
the sequence of the ES-P is shown as SEQ ID NO. 6, wherein the 5 'end is marked with HEX fluorescent reporter gene, and the 3' end is marked with BHQ1 fluorescent quenching gene.
2. A real-time fluorescence quantitative detection pig whole blood toxoplasmosis and eperythrozoon PCR kit is characterized in that: comprising the primer probe set of claim 1, a fluorescent PCR reaction solution, and a positive control standard.
3. The real-time fluorescence quantitative detection PCR kit for toxoplasma gondii and eperythrozoon of pig according to claim 2, characterized in that: the positive control standard comprises a toxoplasma gondii 529 standard and a eperythrozoon 16SrRNA standard;
the standard toxoplasma 529 is a cloning plasmid pUCm-TG of the gene segment of the toxoplasma 529, and the standard eperythrozoon 16SrRNA is a cloning plasmid pUCm-ES of the gene segment of the eperythrozoon 16 SrRNA.
4. A PCR detection method for real-time fluorescence quantitative detection of toxoplasma gondii and eperythrozoon in swine, which is not aimed at diagnosis and treatment of diseases, characterized in that: the dual fluorescent real-time quantitative PCR kit of claim 2 or 3 is used for real-time fluorescent quantitative detection;
the 25 μ L quantitative PCR reaction system included: the upper and lower primers of toxoplasma gondii and eperythrozoon are 0.75 μ L each, and the final concentration is 0.3 μ M; the specific probes for the two viruses are 0.5 mu L respectively, and the final concentration is 0.2 mu M;
the PCR reaction conditions in the detection method are as follows: pre-denaturation at 95 ℃ for 1 min; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 15s, collecting fluorescence signals, and finishing the reaction after 40 cycles.
5. The PCR detection method for real-time fluorescent quantitative determination of toxoplasma hyoscmi and eperythrozoon according to claim 4, wherein the conditions for determining the validity of the detection result are as follows:
(1) detecting no Ct value by a negative control FAM channel and a HEX channel;
(2) the Ct value detected by a positive control FAM channel and a positive control HEX channel is less than or equal to 30.
6. The PCR detection method for real-time fluorescence quantitative determination of toxoplasma hyoscyami and eperythrozoon according to claim 5, wherein the determination criteria of the detection result are:
a. detecting that the Ct value is less than or equal to 40 by both FAM and HEX channels, and judging that the sample is toxoplasma gondii and eperythrozoon mixed infection;
b. the Ct value of the FAM channel is less than or equal to 40, the HEX channel has no Ct value, and the sample is judged to be toxoplasma infection positive and eperythrozoon infection negative;
c. the FAM channel has no Ct value, the HEX channel Ct value is less than or equal to 40, and the sample is judged to be toxoplasma infection negative and eperythrozoon infection positive;
d. and (3) the FAM channel and the HEX channel have no Ct value in detection, and the sample is judged to be negative to toxoplasma gondii and eperythrozoon infection.
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Application publication date: 20211012 |