CN117344058A - PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method - Google Patents
PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method Download PDFInfo
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Abstract
The invention belongs to the technical fields of animal virology and molecular biology, and particularly relates to a PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method, which is a multiplex TaqMan fluorescent quantitative RT-PCR detection method for simultaneously detecting PEDV, PEAV, PDCoV and TGEV four porcine enteroviruses. The invention successfully establishes a multiplex TaqMan fluorescent quantitative PCR detection method for simultaneously distinguishing TGEV, PEDV, PDCoV viruses and PEAV viruses. The method has high stability and specificity, and can provide a powerful tool for health management of pig farming. In addition, the detection method can be applied to virus epidemiological investigation and epidemic situation monitoring, and provides an important reference for controlling and preventing the porcine virus infection.
Description
Technical Field
The invention belongs to the technical fields of animal virology and molecular biology, and particularly relates to a PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method.
Background
In recent years, infectious diarrhea disease is very common in piglets, which is one of the main causes of death of piglets, and causes extremely serious economic loss to the pig industry. Among them, porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), porcine transmissible gastroenteritis virus (Porcine transmissible gastroenteritis virus, TGEV), porcine delta coronavirus (Porcine deltacoronavirus, PDCoV) and porcine entero alpha coronavirus (Porcine enteric alphacoronavirus, PEAV) are the most major pathogens. The 4 viruses can cause high-level infectious intestinal diseases, often cause mixed infection, are difficult to distinguish clinically, mainly cause vomiting, severe diarrhea and death of piglets with the age of less than one week, and therefore, early diagnosis has important significance for preventing and treating the diseases.
Since the clinical symptoms and pathological changes of 4 viruses are very similar, it is difficult to distinguish the pathogens only by clinical diagnosis, and thus detection by laboratory methods is required. Although separation identification and immune related detection means are already applied to virus diagnosis, the prior related technology cannot be used for efficient and rapid pathogen diagnosis due to limitations of detection conditions, time, accuracy and other factors. Compared with the conventional PCR method, the method can only carry out qualitative detection on pathogens, is difficult to accurately quantify, and can realize efficient DNA quantitative and qualitative analysis by fluorescent quantitative PCR. Some scholars at home and abroad have established a fluorescent quantitative RT-PCR identification detection method aiming at one or more viruses in PEDV, PEAV, PDCoV, TGEV. However, there is no method for simultaneous detection and differentiation of PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative RT-PCR detection.
Disclosure of Invention
The invention provides a PEDV, PEAV, PDCoV, TGEV multiple TaqMan fluorescent quantitative detection method, which can detect PEDV, PEAV, PDCoV and TGEV four porcine enteroviruses simultaneously, provides important experimental technical support for early detection and prevention and control of porcine diarrhea viruses in the future and provides a new technical means for development of diagnostic kits.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a PEDV, PEAV, PDCoV, TGEV multiple TaqMan fluorescent quantitative detection method, which is a multiple TaqMan fluorescent quantitative RT-PCR detection method for simultaneously detecting PEDV, PEAV, PDCoV and TGEV four porcine enteroviruses;
the TaqMan fluorescence quantitative detection method comprises the following steps:
sample treatment: taking a diarrhea sample to be detected, adding a PBS solution, oscillating, centrifuging, and taking a supernatant for later use;
extraction of genome: extracting total RNA from the obtained supernatant, and then carrying out reverse transcription to obtain cDNA;
multiplex TaqMan fluorescent quantitative RT-PCR amplification reaction: taking the obtained cDNA as a detection template, carrying out multiplex TaqMan fluorescent quantitative reaction amplification with a PEDV, PEAV, PDCoV, TGEV virus specific amplification primer and a corresponding TaqMan fluorescent probe under the condition of a reaction system, and collecting fluorescent signals;
and (3) result detection: the result determination is performed by Ct value.
Preferably, the conditions of centrifugation are: centrifuging at 4000-9000 r/min for 4-12 min.
Preferably, the diarrhea sample to be tested comprises an intestinal tissue sample, a fecal swab sample and an anal swab sample of diarrhea pigs.
Preferably, the method for processing the intestinal tissue sample comprises the following steps: the sample processing method of the intestinal tissue sample comprises the following steps: taking diseased intestinal tissues, adding the diseased intestinal tissues into a sterilizing centrifuge tube containing PBS solution, grinding in a grinder, centrifuging for 4-6 min at 7000-9000 r/min, and taking supernatant for later use.
Preferably, the sample processing method of the fecal swab sample comprises the following steps: taking a fecal sample, adding the fecal sample into a sterilizing centrifuge tube containing PBS solution, repeatedly oscillating for 2 times, centrifuging for 9-11 min at 7000-9000 r/min, and taking supernatant for later use;
preferably, the sample processing method of the anal swab sample comprises the following steps: adding the anus swab sample into a sterilizing centrifuge tube containing PBS solution, repeatedly oscillating for 2 times, centrifuging for 9-11 min at 4000-6000 r/min, and taking supernatant for later use.
Preferably, the specific amplification primers of PEDV comprise an upstream primer and a downstream primer; the specific amplification primers of the PEAV comprise an upstream primer and a downstream primer; the specific amplification primers of the PDCoV comprise an upstream primer and a downstream primer; the specific amplification primers of the TGEV comprise an upstream primer and a downstream primer.
Preferably, the upstream primer in the specific amplification primer of the PEDV is PEDV-JD180-F, the downstream primer is PEDV-JD180-R, and the corresponding TaqMan fluorescent probe is PEDV-P; the nucleotide sequence of the PEDV-JD180-F is shown as SEQ ID NO.1, the nucleotide sequence of the PEDV-JD180-R is shown as SEQ ID NO.2, and the nucleotide sequence of the PEDV-P is shown as SEQ ID NO. 3;
the upstream primer in the specific amplification primer of the PEAV is PEAV-JD120-F, the downstream primer is PEAV-JD120-R, and the corresponding TaqMan fluorescent probe is PEAV-P; the nucleotide sequence of the PEAV-JD120-F is shown as SEQ ID NO.4, the nucleotide sequence of the PEAV-JD120-R is shown as SEQ ID NO.5, and the nucleotide sequence of the PEAV-P is shown as SEQ ID NO. 6;
the upstream primer in the PDCoV specific amplification primer is PDCoV-JD135-F, the downstream primer is PDCoV-JD135-R, and the corresponding TaqMan fluorescent probe is PDCoV-P; the nucleotide sequence of the PDCoV-JD135-F is shown as SEQ ID NO.7, the nucleotide sequence of the PDCoV-JD135-R is shown as SEQ ID NO.28, and the nucleotide sequence of the PDCoV-P is shown as SEQ ID NO. 9;
the upstream primer in the specific amplification primer of the TGEV is TGEV-JD143-F, the downstream primer is TGEV-JD-143-R, and the corresponding TaqMan fluorescent probe is TGEV-P; the nucleotide sequence of the TGEV-JD143-F is shown as SEQ ID NO.10, the nucleotide sequence of the TGEV-JD-143-R is shown as SEQ ID NO.11, and the nucleotide sequence of the TGEV-P is shown as SEQ ID NO. 12.
Preferably, the amplification reaction system solution during the amplification of the multiplex TaqMan fluorescent quantitative reaction specifically comprises the following components in volume: 2X Animal DetectionU Probe MasterMix: 12.5. Mu.l each of the PEDV upstream and downstream primers, 0.5. Mu.l each of the PEAV upstream and downstream primers, 0.5. Mu.l each of the PDCoV upstream and downstream primers, and 0.5. Mu.l each of the TGEV upstream and downstream primers; PEDV probe 0.2. Mu.l, PDCoV probe 0.2. Mu.l, PEAV probe 0.5. Mu.l, TGEV probe 0.5. Mu.l, template DNA 2. Mu.l, and the system was made up to 25. Mu.l by adding double distilled water.
Preferably, the reaction program for amplifying the multiplex TaqMan fluorescent quantitative reaction is as follows: pre-denaturation at 95 ℃ for 30s; the cycling reactions included 95℃10s,60℃30s, 45 cycles total, and fluorescence signal collection at the last second setting at 60 ℃.
Preferably, the PBS solution has a pH of 7.1 to 7.3 and a concentration of 0.1mol/L.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method which can detect four porcine enteroviruses including porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), porcine enterovirus alpha (Porcine enteric alphacoronavirus, PEAV), porcine delta coronavirus (Porcine deltacoronavirus, PDCoV) and porcine transmissible gastroenteritis virus (Porcine transmissible gastroenteritis virus, TGEV) simultaneously. The invention designs specific primers and probes aiming at the gene sequences of the M gene of PEDV, the N gene of PEAV, the M gene of PDCoV and the N gene of TGEV, researches reaction conditions, establishes a multiplex fluorescence quantitative PCR identification detection method capable of rapidly detecting and identifying the 4 diseases, provides important experimental technical support for early detection and prevention and control of porcine diarrhea viruses in the future, and provides a new technical means for the development of diagnostic kits.
The detection method established by the invention only specifically amplifies PEDV, PEAV, PDCoV and TGEV, has no cross reaction with other related viruses, and has stronger specificity. The detection sensitivity of the detection method to PEDV, PEAV, PDCoV, TGEV is respectively 30.2copies/L, 23.8copies/L, 33.4copies/L, 28.6copies/L and PEDV, PEAV, PDCoV, TGEV, the CV maximum values of the repeated tests in the groups are respectively 1%, 0.8%, 0.6% and 0.5%, and the CV maximum values of the repeated tests in the groups are respectively 1%, 0.7% and 0.8% and are not more than 1%, which indicates that the established method has good repeatability; the PRoV, PRRSV, CSFV, PRV, PCV virus sample is detected by the method, and no cross reaction exists, which shows that the detection method has good specificity. Meanwhile, the established detection method is adopted to detect 231 diarrhea samples clinically, and the result shows that the positive detection rate of PEDV, PEAV, TGEV, PDCoV is 51.52%, 1.73%, 8.66% and 4.76% respectively; wherein the mixed infection rate of PEDV and TGEV is 1.3%; the mixed infection rate of PEDV and PEAV is 0.43%; the mixed infection rate of PEDV and PDCoV is 0.43%; the mixed infection rate of TGEV and PDCoV is 0.43%; the mixed infection rate of PEDV and TGEV is 1.3%; the mixed infection rate of PEDV and PEAV is 0.43%; the coincidence rate of both PCR was 88% compared with ordinary PCR. The detection method established by the invention provides a rapid, specific, sensitive and efficient technical means for the identification detection of PEDV, PEAV, PDCoV and TGEV and epidemiological investigation.
Drawings
FIG. 1 is a PEAV fluorescence quantitative RT-PCR standard curve prepared by a standard curve of a recombinant plasmid standard in example 1 of the present invention, wherein A is a standard curve of PEDV; b in the figure is a PEAV standard curve; c is the standard curve of PDCoV; in the graph, D is the standard curve of TGEV.
FIG. 2 is a graph showing the sensitivity test results in example 1 of the present invention, wherein A is a standard curve of PEDV; b in the figure is a PEAV standard curve; c is the standard curve of PDCoV; in the graph, D is the standard curve of TGEV; 1 to 10 each represent: dilution of 10 -1 ～10^ -10 Is a mixed standard plasmid of (2); 11 is: negative control.
FIG. 3 shows the results of the specificity test of example 1 of the present invention, wherein A is the standard curve of PEDV; b in the figure is a PEAV standard curve; c is the standard curve of PDCoV; in the graph, D is the standard curve of TGEV; 1 is: mixing standard plasmids; 2 is: PEDV;3 is: PEAV;4 is: TGEV;5 is: PDCoV;6 to 10 are respectively: PRoV, PRRSV, CSFV, PRV, PCV2;11 is: negative control.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
The following are the samples and reagents used in the examples:
(1) Sample source
Positive samples and nucleic acids of Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV), porcine delta coronavirus (PDCoV), porcine entero-alpha coronavirus porcine reproductive (PEAV), respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV 2), porcine circovirus type 3 (PCV 3), swine fever virus (CSFV), porcine rotavirus (podv), pseudorabies virus (PRV) were all stored in fowls livestock infectious disease control and biotechnology focus laboratory.
The diarrhea samples to be tested used in the examples were: intestinal tissue, fecal swab and anal swab samples of diarrhea piglets submitted by pig farms in different areas of Fujian province were collected in 2016-2022.
(2) Main reagent
2X Animal Detection U Probe MasterMix, 2X Taq Plus MasterMix II, hiScript III 1st Strand cDNA Synthesis Kit were all purchased from Nanjinozan Biotechnology Co.
pMD19-T vector, DL500 DNAMaroer, etc. are all available from Takara doctor materials technologies Inc.
DH 5. Alpha. Competent cells were purchased from Shanghai Biotechnology Inc.
The gel recovery kit is from Tiangen Biochemical technology Co.
Example 1
The construction of the PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method comprises the following steps:
1. primer design and Synthesis
Specific amplification primers for the 4 pairs of viruses and corresponding TaqMan fluorescent probes are designed respectively by referring to the M gene sequence of PEDV, the M gene sequence of PEAV, the M gene sequence of PDCoV and the N gene sequence of TGEV in GenBank. Specific amplification primers for the 4 viruses and corresponding TaqMan fluorescent probe sequences are shown in Table 1.
TABLE 1PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative RT-PCR primer and probe sequences
2. Sample processing
Different diarrhea samples are treated by different treatment methods, and the specific steps are as follows:
(1) Fecal sample: 200mg of feces are taken and added into a sterilizing centrifuge tube containing lmLPBS solution, the mixture is repeatedly oscillated for 2 times, centrifuged for 10min at 5000r/min, and the supernatant is taken for standby. Wherein, PBS solution is purchased from Shanghai source culture Biotech Co., ltd., product number: b310KJ. The PBS solution had a pH of 7.2 and a concentration of 0.1mol/L.
(2) Intestinal tissue sample: 50mg of diseased intestinal tissue is taken, added into a sterilizing centrifuge tube containing 1mLPBS solution, ground in a grinder, centrifuged at 8000r/min for 5min, and the supernatant is taken for later use. Wherein, PBS solution is purchased from Shanghai source culture Biotech Co., ltd., product number: b310KJ. The PBS solution had a pH of 7.2 and a concentration of 0.1mol/L.
(3) Anal swab sample: adding the anus swab sample into a sterilizing centrifuge tube containing lmLPBS solution, repeatedly oscillating for 2 times, centrifuging at 5000r/min for 10min, and collecting supernatant for later use. Wherein, PBS solution is purchased from Shanghai source culture Biotech Co., ltd., product number: b310KJ. The PBS solution had a pH of 7.2 and a concentration of 0.1mol/L.
And after the different diarrhea samples are treated, respectively taking the supernatant after the centrifugation of the diarrhea samples, extracting total RNA in the supernatant of the different diarrhea samples according to the specification of an RNA/DNA extraction kit, carrying out reverse transcription to obtain cDNA, and preserving at-20 ℃ for later use. And respectively extracting different diarrhea samples to obtain total RNA. Wherein, the RNA/DNA extraction kit is a DNA/RNA membrane column extraction kit of 50T sequencing grade, purchased from Iceland Ensate Biotechnology Co.
3. Preparation of recombinant plasmid Standard
Taking cDNA obtained by extracting total RNA from the intestinal tissue sample and then carrying out reverse transcription, and respectively carrying out PCR amplification reaction by using specific amplification primers corresponding to 4 viruses and corresponding TaqMan fluorescent probes in the table 1 by taking the cDNA as a template.
Namely, the PEDV is amplified by PCR by using the specific amplification primers corresponding to the PEDV and the corresponding TaqMan fluorescent probes in the table 1, and thus a PEDV M gene (180 bp) fragment is obtained. Wherein the specific amplification primers of PEDV comprise an upstream primer and a downstream primer. The upstream primer in the specific amplification primer of the PEDV is PEDV-JD180-F, the downstream primer is PEDV-JD180-R, and the corresponding TaqMan fluorescent probe is PEDV-P; the nucleotide sequence of the PEDV-JD180-F is shown as SEQ ID NO.1, the nucleotide sequence of the PEDV-JD180-R is shown as SEQ ID NO.2, and the nucleotide sequence of the PEDV-P is shown as SEQ ID NO. 3.
Namely, the PEAV is amplified by PCR by using the specific amplification primers corresponding to the PEAV and the corresponding TaqMan fluorescent probes in the table 1, and the PEAVN gene (120 bp) fragment is obtained. Wherein, the specific amplification primers of PEAV comprise an upstream primer and a downstream primer. The upstream primer in the specific amplification primer of the PEAV is PEAV-JD120-F, the downstream primer is PEAV-JD120-R, and the corresponding TaqMan fluorescent probe is PEAV-P; the nucleotide sequence of PEAV-JD120-F is shown as SEQ ID NO.4, the nucleotide sequence of PEAV-JD120-R is shown as SEQ ID NO.5, and the nucleotide sequence of PEAV-P is shown as SEQ ID NO. 6.
Namely, the PDCoV is amplified by PCR by using the specific amplification primers corresponding to the PDCoV and the TaqMan fluorescent probes corresponding to the specific amplification primers in the table 1, so that a PDCoV M gene (135 bp) fragment is obtained. Specific amplification primers for PDCoV include an upstream primer and a downstream primer. The upstream primer in the specific amplification primer of the PDCoV is PDCoV-JD135-F, the downstream primer is PDCoV-JD135-R, and the corresponding TaqMan fluorescent probe is PDCoV-P; the nucleotide sequence of PDCoV-JD135-F is shown as SEQ ID NO.7, the nucleotide sequence of PDCoV-JD135-R is shown as SEQ ID NO.28, and the nucleotide sequence of PDCoV-P is shown as SEQ ID NO. 9.
Namely, the TGEV is subjected to PCR amplification by using the specific amplification primers corresponding to the TGEV and the corresponding TaqMan fluorescent probes in the table 1, so as to obtain a TGEV N gene (143 bp) fragment. Specific amplification primers for TGEV include an upstream primer and a downstream primer. The upstream primer in the specific amplification primer of the TGEV is TGEV-JD143-F, the downstream primer is TGEV-JD-143-R, and the corresponding TaqMan fluorescent probe is TGEV-P; the nucleotide sequence of TGEV-JD143-F is shown as SEQ ID NO.10, the nucleotide sequence of TGEV-JD-143-R is shown as SEQ ID NO.11, and the nucleotide sequence of TGEV-P is shown as SEQ ID NO. 12.
After the reaction is finished, respectively taking PCR amplified products corresponding to 4 viruses, carrying out agarose gel electrophoresis, recovering and purifying by using a gel-cut recovery kit, respectively connecting the purified products with a pMD19-T carrier, converting the pMD19-T carrier into DH5 alpha competent cells, coating bacterial liquid on a LA culture medium for overnight culture for 16 hours, picking single bacterial colony for identification, extracting plasmids by using a plasmid extraction kit, and sequencing by Shanghai biological engineering Co Ltd. The positive plasmid (recombinant plasmid) with correct sequence is used as recombinant plasmid standard substance, and the prepared 4 recombinant plasmid standard substances are pMD-PEDV, pMD-PEAV, pMD-PDCoV and pMD-TGEV respectively.
Wherein, the formula of the LA culture medium is as follows: tryptone (Tryptone) 10g/L, beef extract (Yeast extract) 5g/L sodium chloride (NaCl) 10g/L, agar 15g/L, ampicillin (AMP) 200ul/L. The preparation method comprises the following steps: according to the formula of culture medium, weighing various raw materials, sequentially adding into conical flask to dissolve, supplementing required water, autoclaving, cooling to below 60deg.C, adding ampicillin, preparing plate culture medium in ultra clean bench, cooling, solidifying, and storing in refrigerator at 4deg.C.
The concentration of the recombinant plasmid standard was measured with an ultramicro nucleic acid protein meter and its copy number was calculated.
Wherein, standard plasmid copy number (copies/. Mu.L) = (6.02X10) 23 ) X [ Standard quality pellet concentration (ng/. Mu.L). Times.10 ] -9 ]/(DNAlength×660)。
The concentration detection concentrations of the recombinant plasmid standard substances measured by the ultramicro nucleic acid protein determinator are respectively; the detection concentration of pMD-PEDV was 122.6 ng/. Mu.L, that of pMD-PEAV was 88.3 ng/. Mu.L, that of pMD-PDCoV was 120.8 ng/. Mu.L, and that of pMD-TGEV was 102.7 ng/. Mu.L. The conversion to copy number is as follows: pMD-PEDV: 3.02X10 10 copies/μL，pMD-PEAV：2.38×10 10 copies/μL，pMD-PDCoV：3.34×10 10 copies/μL，pMD-TGEV：2.86×10 10 copies/μL。
4. Reaction procedure
The 4 recombinant plasmid standard products constructed above are used as templates, the specific amplification primers corresponding to the 4 viruses in the table 1 and the corresponding TaqMan fluorescent probes are respectively used for multiplex TaqMan fluorescent quantitative PCR amplification in the same reaction system, and the annealing temperature, the primer concentration and the probe concentration are respectively researched to obtain the optimal reaction conditions of the TaqMan fluorescent quantitative PCR.
The annealing temperature, primer and probe concentration were performed to determine the amplification conditions. The specific studies were as follows: with reference to the reaction conditions provided by the instructions of the fluorescent quantitative PCR reagent of Takara corporation, and the selected gene segment of interest, the main conditions affecting the fluorescent quantitative PCR were studied, including primer concentration, probe concentration and annealing temperature. Wherein the levels of the primer addition were 0.5ul, 0.8ul and 1.0ul, respectively, the levels of the probe addition were 0.2ul, 0.4ul and 0.5ul, and the annealing temperature was three gradients of 58 ℃, 59 ℃ and 60 ℃, respectively, and the reaction conditions were studied. Among them, the fluorescent quantitative PCR reagent of Takara company is HiScript III 1st Strand cDNA Synthesis Kit, purchased from Nanjinozan Biotechnology Co., ltd.
The best reaction system obtained by the above experiment is as follows:
25 μl: 2X Animal DetectionU Probe MasterMix:12.5ul of each of the PEDV upstream and downstream primers, 0.5ul of each of the PEAV upstream and downstream primers, 0.5ul of each of the PDCoV upstream and downstream primers, and 0.5ul of each of the TGEV upstream and downstream primers; PEDV probe 0.2ul, pdcov probe 0.2ul, peav probe 0.5ul, tgev probe 0.5ul, template DNA2ul, the system was made up to 25ul by adding double distilled water. The reaction procedure is: pre-denaturation at 95 ℃ for 30s; the cycling reactions included 95℃10s,60℃30s, 45 cycles total, and fluorescence signal collection at the last second setting at 60 ℃.
Experimental example 1
A PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method, comprising the steps of:
(1) Sample treatment: taking a diarrhea sample of a pig to be detected (here, taking an intestinal tissue sample), adding the diarrhea sample into a sterilizing centrifuge tube containing an lmL PBS solution, repeatedly oscillating for 2 times, centrifuging for 5min at 8000r/min, and taking supernatant for later use. Wherein, PBS solution is purchased from Shanghai source culture Biotech Co., ltd., product number: b310KJ. The PBS solution had a pH of 7.2 and a concentration of 0.1mol/L.
It should be noted that different diarrhea samples were treated differently (see example 1). Meanwhile, different diarrhea samples can also be subjected to sample treatment by adopting the treatment method, and specific parameters can be properly adjusted according to actual samples.
(2) Extraction of genome: extracting total RNA from the supernatant obtained in the step (1) by using an RNA/DNA extraction kit (50T sequencing grade of a virus DNA/RNA membrane column method extraction kit of Iceladon biosciences, inc.), and then carrying out reverse transcription to obtain cDNA and storing at-20 ℃ for later use.
(3) Multiplex TaqMan fluorescent quantitative RT-PCR amplification reaction: and (3) taking the cDNA obtained in the step (2) as a detection template, respectively carrying out multiplex TaqMan fluorescent quantitative reaction amplification with the specific amplification primers of 4 viruses in the table 1 and the corresponding TaqMan fluorescent probes under the optimal reaction system condition, and collecting fluorescent signals.
In step (3), a virus is tested once during the PCR amplification reaction. Namely, performing PCR amplification on PEDV by using the specific amplification primers corresponding to the PEDV and the corresponding TaqMan fluorescent probes in table 1; specific amplification primers for PEDV include an upstream primer and a downstream primer. The upstream primer in the specific amplification primer of the PEDV is PEDV-JD180-F, the downstream primer is PEDV-JD180-R, and the corresponding TaqMan fluorescent probe is PEDV-P.
Namely, carrying out PCR amplification on PEAV by utilizing the specific amplification primers corresponding to the PEAV and the TaqMan fluorescent probes corresponding to the specific amplification primers in table 1; specific amplification primers for PEAV include an upstream primer and a downstream primer. The upstream primer in the specific amplification primer of the PEAV is PEAV-JD120-F, the downstream primer is PEAV-JD120-R, and the corresponding TaqMan fluorescent probe is PEAV-P.
Namely, performing PCR amplification on the PDCoV by using the specific amplification primers corresponding to the PDCoV in the table 1 and the corresponding TaqMan fluorescent probes; specific amplification primers for PDCoV include an upstream primer and a downstream primer. The upstream primer in the PDCoV specific amplification primer is PDCoV-JD135-F, the downstream primer is PDCoV-JD135-R, and the corresponding TaqMan fluorescent probe is PDCoV-P.
Namely, performing PCR amplification on TGEV by using the specific amplification primers corresponding to the TGEV and the corresponding TaqMan fluorescent probes in table 1; the upstream primer in the specific amplification primer of the TGEV is TGEV-JD143-F, the downstream primer is TGEV-JD-143-R, and the corresponding TaqMan fluorescent probe is TGEV-P.
(4) And (3) result detection: drawing an amplification curve, reading a corresponding Ct value and judging a result; the result judging method is that according to the amplification curve and the Ct value, the result can be judged to be positive when the Ct value is less than or equal to 36 cycles, otherwise, the result is judged to be negative.
The invention respectively carries out sensitivity, specificity and repeatability test analysis on an established PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescence quantitative detection method, and the specific test process is as follows:
(1) Standard curve drawing
Recombinant plasmid standard substances of 4 viruses are respectively subjected to 10-time gradient dilution, and then dilution is selected to be 10 -1 ～10 -10 And (3) performing multiplex TaqMan fluorescent quantitative reaction amplification under the optimal reaction system condition as a template, and drawing a standard curve which is shown in figure 1. A recombinant plasmid standard of virus is reacted once. Wherein, the 4 recombinant plasmid standards are respectively: the recombinant plasmid standard of PEDV is pMD-PEDV, the recombinant plasmid standard of PEAV is pMD-PEAV, the recombinant plasmid standard of PDCoV is pMD-PDCoV, and the recombinant plasmid standard of TGEV is pMD-TGEV.
Wherein, PEAV standard curve: correlation coefficient R 2 =0.998, amplification efficiency e=1;
TGEV standard curve: correlation coefficient R 2 =0.998, amplification efficiency e=1.06;
PEDV standard curve: correlation coefficient R 2 =0.994, amplification efficiency e=1.09;
standard curve: correlation coefficient R 2 =0.998, amplification efficiency e=1.05.
The standard curve can be used for obtaining that the detection PEDV, PEAV, PDCoV, TGEV four-channel multiplex TaqMan fluorescent quantitative PCR has a good linear relationship.
(2) Sensitivity test
An equal proportion of the mixture of the recombinant plasmid standard products of 4 viruses after 10-time gradient dilution is used as a template, and the detection method established according to the example 1 is respectively applied to detection, and a standard curve is established while the lowest detectable concentration is determined. The detection results are shown in FIG. 2.
As can be seen from FIG. 2, the dilution is 10 -9 The amplification curve still appears in the mixed standard plasmid detection of (2), but the CT value is detected>37 and the stability is poor, so that the detection method established in the study is judged to have the lowest detection amount of 30.2 copies/mu L of PEDV recombinant standard plasmid, 23.8 copies/mu L of PEAV recombinant standard plasmid, 33.4 copies/mu L of PDCoV recombinant standard plasmid and 28.6 copies/mu L of TGEV recombinant standard plasmid respectively.
(3) Specificity test
The virus solution containing PRoV, PRRSV, CSFV, PRV, PCV is extracted by using a Tsingtao Ensat Biotechnology Co., ltd, a virus DNA/RNA membrane column method extraction kit 50T sequencing grade kit, and then is inverted into cDNA, the cDNA and the DNA are used as templates, a recombinant plasmid standard is used as a positive control, double distilled water is used as a negative control, and the detection is carried out by using the detection method established according to the embodiment 1, and the detection result is shown in figure 3.
As can be seen from FIG. 3, the FAM channel has no amplification curve except the amplification curve of the mixed standard plasmid and PEDV; the HEX channel has no amplification curve except the mixed plasmid and PEAV; the ROX channel has no amplification curve except the mixed plasmid and TGEV; the CY5 channel has no amplification curve except the amplification curve of the mixed plasmid and the PDCoV. The established detection method is proved to have good specificity.
(4) Repeatability test
Recombinant plasmid standard substances of 4 viruses are respectively processed according to 10 -2 、10 -4 、10 -6 After dilution, the mixed standard plasmids with each concentration are uniformly mixed to be used as templates, and the repeatability test is carried out by respectively applying the detection method established according to the embodiment 1, and three repeated verification intra-group variation coefficients are set in the same batch to prepare three batch verification inter-group variation coefficients. The results are shown in tables 2 and 3.
TABLE 2 within-batch repeatability test results of PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method
TABLE 3 results of batch-to-batch reproducibility test of PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative determination methods
Wherein, the repeated test CV value in the TGEV group is between 0.3 and 0.5 percent, and the repeated test CV value in the group is between 0.7 and 0.8 percent; the repeated test CV value in the PEDV group is between 0.4 and 1 percent, and the repeated test CV value between groups is between 0.5 and 1 percent; the repeated test CV value in the PEAV group is between 0.5 and 0.8 percent, the repeated test CV value in the PDCoV group is between 0.4 and 1 percent, the repeated test CV value in the PDCoV group is between 0.5 and 0.6 percent, and the repeated test CV value in the PEAV group is between 0.4 and 0.7 percent, which are all less than 1 percent, thus indicating that the stability and the repeatability of the multiplex fluorescence quantitative PCR are better.
In order to verify the PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method established by the invention, the following clinical sample detection test is carried out:
231 diarrhea samples, including intestinal tissue, fecal tissue, and anal swab from sick pigs, were collected from a large-scale pig farm with diarrhea occurring in different areas 2016-2022 of Fujian. The PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method established according to the invention is used for detection and analysis, meanwhile, the common RT-PCR detection method is used for detecting 231 diarrhea samples, the detection PEDV, TGEV, PDCoV, PEAV results of the two samples are compared, and the coincidence rate of the two samples is calculated, wherein the specific result is shown in Table 4.
Table 4PEDV, PEAV, PDCoV, TGEV multiple TaqMan fluorescent quantitative determination method clinical sample detection results
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The coincidence rate of the positive results of the detected samples by the two methods of calculation and analysis is 88 percent. The PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescence quantitative detection method established by the research can be used for clinical detection, and the pathogenic staggering and complicating of diarrhea caused by large-scale pig farms in Fujian province are demonstrated, and attention should be paid.
From the experimental results, the PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method provided by the invention is more sensitive and better in effect when being used for detecting PEDV, PEAV, PDCoV, TGEV four pig enteroviruses than when being used for detecting PEDV, PEAV, PDCoV, TGEV four pig enteroviruses by a common RT-PCR detection method.
The invention provides a PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method which can detect four porcine enteroviruses including porcine epidemic diarrhea virus (Porcine epidemic diarrhea virus, PEDV), porcine enterovirus alpha (Porcine enteric alphacoronavirus, PEAV), porcine delta coronavirus (Porcine deltacoronavirus, PDCoV) and porcine transmissible gastroenteritis virus (Porcine transmissible gastroenteritis virus, TGEV) simultaneously. The invention designs specific primers and probes aiming at the gene sequences of the M gene of PEDV, the N gene of PEAV, the M gene of PDCoV and the N gene of TGEV, researches reaction conditions, establishes a multiplex fluorescence quantitative PCR identification detection method capable of rapidly detecting and identifying the 4 diseases, provides important experimental technical support for early detection and prevention and control of porcine diarrhea viruses in the future, and provides a new technical means for the development of diagnostic kits.
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. A PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method is characterized in that the method is a multiplex TaqMan fluorescent quantitative RT-PCR detection method for simultaneously detecting PEDV, PEAV, PDCoV and TGEV four porcine enteroviruses;
the TaqMan fluorescence quantitative detection method comprises the following steps:
sample treatment: taking a diarrhea sample to be detected, adding a PBS solution, oscillating, centrifuging, and taking a supernatant for later use;
extraction of genome: extracting total RNA from the obtained supernatant, and then carrying out reverse transcription to obtain cDNA;
multiplex TaqMan fluorescent quantitative RT-PCR amplification reaction: taking the obtained cDNA as a detection template, carrying out multiplex TaqMan fluorescent quantitative reaction amplification with a PEDV, PEAV, PDCoV, TGEV virus specific amplification primer and a corresponding TaqMan fluorescent probe under the condition of a reaction system, and collecting fluorescent signals;
and (3) result detection: the result determination is performed by Ct value.
2. The PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method according to claim 1, wherein the centrifugation conditions are as follows: centrifuging at 4000-9000 r/min for 4-12 min.
3. The PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method of claim 1, wherein the diarrhea sample comprises intestinal tissue sample, fecal swab sample and anal swab sample of diarrhea pigs.
4. The method for quantitative detection of PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescence according to claim 3, wherein the method for processing the intestinal tissue sample comprises the steps of: taking diseased intestinal tissues, adding the diseased intestinal tissues into a sterilizing centrifuge tube containing PBS solution, grinding in a grinder, centrifuging for 4-6 min at 7000-9000 r/min, and taking supernatant for later use.
5. A PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative assay according to claim 3, wherein the sample processing method of the fecal swab sample comprises the steps of: taking a fecal sample, adding the fecal sample into a sterilizing centrifuge tube containing PBS solution, repeatedly oscillating for 2 times, centrifuging for 9-11 min at 7000-9000 r/min, and taking supernatant for later use.
6. The method for quantitative detection of PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescence according to claim 3, wherein the method for processing the sample of anal swab sample comprises the following steps: adding the anus swab sample into a sterilizing centrifuge tube containing PBS solution, repeatedly oscillating for 2 times, centrifuging for 9-11 min at 4000-6000 r/min, and taking supernatant for later use.
7. The method for quantitative detection of PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescence according to claim 5, wherein the upstream primer in the specific amplification primer of PEDV is PEDV-JD180-F, the downstream primer is PEDV-JD180-R, and the corresponding TaqMan fluorescent probe is PEDV-P; the nucleotide sequence of the PEDV-JD180-F is shown as SEQ ID NO.1, the nucleotide sequence of the PEDV-JD180-R is shown as SEQ ID NO.2, and the nucleotide sequence of the PEDV-P is shown as SEQ ID NO. 3;
the upstream primer in the specific amplification primer of the PEAV is PEAV-JD120-F, the downstream primer is PEAV-JD120-R, and the corresponding TaqMan fluorescent probe is PEAV-P; the nucleotide sequence of the PEAV-JD120-F is shown as SEQ ID NO.4, the nucleotide sequence of the PEAV-JD120-R is shown as SEQ ID NO.5, and the nucleotide sequence of the PEAV-P is shown as SEQ ID NO. 6;
the upstream primer in the PDCoV specific amplification primer is PDCoV-JD135-F, the downstream primer is PDCoV-JD135-R, and the corresponding TaqMan fluorescent probe is PDCoV-P; the nucleotide sequence of the PDCoV-JD135-F is shown as SEQ ID NO.7, the nucleotide sequence of the PDCoV-JD135-R is shown as SEQ ID NO.28, and the nucleotide sequence of the PDCoV-P is shown as SEQ ID NO. 9;
the upstream primer in the specific amplification primer of the TGEV is TGEV-JD143-F, the downstream primer is TGEV-JD-143-R, and the corresponding TaqMan fluorescent probe is TGEV-P; the nucleotide sequence of the TGEV-JD143-F is shown as SEQ ID NO.10, the nucleotide sequence of the TGEV-JD-143-R is shown as SEQ ID NO.11, and the nucleotide sequence of the TGEV-P is shown as SEQ ID NO. 12.
8. The PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescent quantitative detection method according to claim 1, wherein the amplification reaction system solution for amplification of the multiplex TaqMan fluorescent quantitative reaction specifically comprises the following components in volume: 2X Animal Detection U Probe Master Mix: 12.5. Mu.l each of the PEDV upstream and downstream primers, 0.5. Mu.l each of the PEAV upstream and downstream primers, 0.5. Mu.l each of the PDCoV upstream and downstream primers, and 0.5. Mu.l each of the TGEV upstream and downstream primers; PEDV probe 0.2. Mu.l, PDCoV probe 0.2. Mu.l, PEAV probe 0.5. Mu.l, TGEV probe 0.5. Mu.l, template DNA 2. Mu.l, and the system was made up to 25. Mu.l by adding double distilled water.
9. The method for quantitative detection of PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescence according to claim 1, wherein the reaction procedure for amplification of the multiplex TaqMan fluorescence quantitative reaction is as follows: pre-denaturation at 95 ℃ for 30s; the cycling reactions included 95℃10s,60℃30s, 45 cycles total, and fluorescence signal collection at the last second setting at 60 ℃.
10. The method for quantitative detection of PEDV, PEAV, PDCoV, TGEV multiplex TaqMan fluorescence according to claim 1, wherein the PBS solution has a pH of 7.1-7.3 and a concentration of 0.1mol/L.
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