CN114703179A - RT-RAA-LFD primer pair, probe, test strip, kit for detecting PDCoV and application thereof - Google Patents
RT-RAA-LFD primer pair, probe, test strip, kit for detecting PDCoV and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of animal epidemic disease detection, in particular to an RT-RAA-LFD primer pair, a probe, a test strip, a kit and application thereof for detecting PDCoV. The invention takes ORF1b gene of PDCoV as a diagnosis target, designs, screens and optimizes to finally obtain a primer pair and a probe which can specifically detect PDCoV nucleic acid. The primer pair, the probe, the kit and the test strip can realize the rapid detection of PDCoV, and the result obtained by isothermal amplification is directly interpreted after being detected by the nucleic acid detection test strip. The whole detection process does not need expensive instrument and equipment, and the detection result can be obtained only through visual interpretation. The invention has wide application prospect in the aspects of rapid detection and discrimination of PDCoV infection at the basic level and on site.
Description
Technical Field
The invention relates to the technical field of animal epidemic disease detection, and particularly relates to an RT-RAA-LFD primer pair, a probe, a test strip, a kit and application for detecting PDCoV nucleic acid.
Background
PDCoV is a newly discovered porcine intestinal pathogenic coronavirus in recent years, mainly infects newborn piglets, has clinical symptoms of acute diarrhea, vomiting, dehydration, emaciation and the like, and death of severe people, and becomes one of important pathogenies influencing pig production; although pigs of other ages may also be infected, the symptoms are relatively mild. PDCoV can be infected by direct contact of healthy pigs with various secretions (such as feces, vomit, saliva, milk, nasal secretions, semen, etc.) of infected or diseased pigs, or by indirect contact with feeds, appliances, transportation, breeders, etc. contaminated with viruses. In 2012, Woo et al first determined the whole genome sequence of PDCoV from pig manure swabs collected between 2007-2011.
It has been shown that PDCoV, in addition to infecting swine, can infect animals of different species, such as wild birds, chickens, turkeys, cattle, etc., suggesting a potential cross-species transmission risk for the Virus (Zhang J., Virus Research,2016,226: 71-84). In 2021, there was a study to isolate PDCoV from the blood of 3 seashore children with fever and abdominal pain symptoms, indicating that PDCoV has potential public health implications (Lednicky j.a., Nature,2021,600: 133-.
In classification, PDCoV belongs to the order of the nested viruses (Nidovirales), the family of Coronaviridae (Coronaviridae), the genus delta coronavirus (Deltacoronavirus). PDCoV is a enveloped single-stranded positive-strand RNA virus, and the genome structure of the PDCoV is as follows: 5 'UTR-ORF 1a/1b-S-E-M-NS6-N-NS 7-3' UTR, approximately 25kb in total length. Of these, ORF1a and ORF1b encode two polyproteins pp1a and pp1ab, and it is speculated that pp1a and pp1ab are cleaved by the protease encoded by ORF1a/1b into about 16 non-structural proteins, which are mainly involved in viral replication and transcription. As clinical symptoms caused by the PDCoV infection are very similar to important porcine diarrhea-causing viruses such as Porcine Epidemic Diarrhea Virus (PEDV), porcine transmissible gastroenteritis virus (TGEV) and porcine rotavirus (PRoV), the PDCoV infection needs to be diagnosed by means of a laboratory detection technology. At present, molecular biological methods such as RT-PCR, real-time fluorescence quantitative RT-PCR, loop-mediated isothermal amplification (LAMP) and the like, indirect immunofluorescence, virus neutralization tests, indirect ELISA and other serological methods are mainly used for detecting and diagnosing PDCoV infection at home and abroad. The diagnostic targets related to the molecular biological detection methods such as RT-PCR, real-time fluorescence quantitative RT-PCR and LAMP comprise genes such as S, M, N and ORF 1. Although the detection method plays an important role in the detection and diagnosis of PDCoV, the defects of complex operation, long time consumption, high requirements on instrument and equipment, excessively complex primer design and the like generally exist, and the actual requirements of clinical on-site rapid and visual detection on PDCoV infected pigs are difficult to meet.
In recent years, a technology based on Recombinase-mediated isothermal amplification (RAA) is showing good application potential in diagnosis and detection of human and veterinary diseases, and has attracted extensive attention worldwide. For example, a pathogen nucleic acid detection method based on RAA and lateral flow immunochromatography has been successfully applied to rapid diagnosis of novel coronavirus (SARS-CoV-2) and African Swine Fever Virus (ASFV), and has already filed for national invention patent protection. The RAA technology mainly depends on single-stranded DNA binding protein (which is combined with single-stranded DNA to maintain the single-stranded state of the DNA and prevent the DNA from being re-paired to form double-stranded DNA), recombinase (which can form a compound with a primer to promote the primer to be paired with a homologous sequence of template DNA and start DNA replication) and DNA polymerase (which is used for amplification and extension) to complete the amplification of target nucleic acid in a short time (less than or equal to 20min) under the constant temperature condition (37-42 ℃).
As the whole RAA amplification process does not need complex and expensive instrument and equipment, and can be detected only by one portable thermostat even by means of body temperature, the RAA technology is very suitable for being developed and applied to on-site rapid detection. On the basis of meeting the requirement of on-site detection, the invention simultaneously introduces a Lateral flow immunochromatographic technique, combines nucleic acid isothermal amplification with Lateral Flow Dipstick (LFD), and thus realizes on-site rapid visual detection of PDCoV infection.
Disclosure of Invention
The invention aims to provide a method for rapidly and visually detecting PDCoV infection.
More specifically, the invention provides a primer pair, a probe, a test strip, a kit and application thereof for a Reverse transcription recombinase-mediated isothermal amplification (RT-RAA) combined Lateral flow test strip (LFD) method for rapidly and visually detecting Porcine delta coronavirus (PDCoV).
In a first aspect, the invention provides a primer probe combination, wherein the sequences of the primers are shown as SEQ ID NO.3 and SEQ ID NO.16, and the sequence of the probe is shown as SEQ ID NO. 17.
In the primer probe combination provided by the invention, the 5 'end of the probe is modified by biotin, the 32 nd base C is replaced by Tetrahydrofuran (THF), and the 3' end is modified by phosphorylation; and (3) carrying out fluorescent group FAM modification on the 5' end of the reverse primer.
The probe sequence is as follows:
5’-Biotin-GGTCCAGAGTACCGCTGTGAGGAGCCGCTTG[THF]TAAATTAGTAGGAGT-Phosphorylation-3’
the reverse primer sequences are as follows:
5’-FAM-ACGTGTTAGGAACATATTATGATACGCATC-3’。
according to the understanding of the technical personnel in the field, the invention requests to protect the application of the primer-probe combination in preparing a PDCoV detection kit or a detection test strip.
In a second aspect, the invention provides a PDCoV RT-RAA-LFD detection kit, which contains the primer probe combination.
The RT-RAA-LFD detection kit provided by the invention also comprises reaction buffer solution and enzymes. Specifically, the enzymes include reverse transcriptase, recombinase, DNA polymerase, single-stranded DNA binding protein, and endonuclease IV.
The reaction system (25.00 mu L) of the RT-RAA-LFD detection kit provided by the invention comprises: 14.70 mu L of reaction buffer solution A; reaction buffer B1.25. mu.L; 10 pmol/. mu.L of forward primer 1.00. mu.L; 10 pmol/. mu.L reverse primer 1.00. mu.L; 0.30. mu.L of 10 pmol/. mu.L probe; PDCoV RNA template 1.00 u L and no nuclease water 5.75 u L.
In a third aspect, the present invention provides a PDCoV nucleic acid detection test strip for detecting a product amplified by the detection kit, the detection test strip comprising: a sample combination pad, a detection line and a quality control line; the sample binding pad contains colloidal gold labeled streptavidin; the detection line contains a mouse anti-FAM monoclonal antibody; the control line contains biotin coupled to Bovine Serum Albumin (BSA).
Specifically, the test strip provided by the invention is fixed on a PVC (polyvinyl chloride) bottom plate and sequentially comprises a sample pad, a combination pad, an NC (NC) membrane and absorbent paper from left to right; the test strip sample combination pad contains streptavidin marked by colloidal gold, the detection line (T line) contains a monoclonal antibody of mouse anti-FAM, and the quality control line (C line) contains biotin coupled with Bovine Serum Albumin (BSA).
In a fourth aspect, the present invention provides a method for using a PDCoV nucleic acid detection test strip, comprising:
taking RNA extracted from a sample to be detected as a template, amplifying by using the detection kit to obtain an amplification product, and performing visual interpretation on the amplification product by using the detection test strip;
the result judgment standard is as follows: two red strips appear in the positive result, one is positioned on a detection line (T line), and the other is positioned on a quality control line (C line); negative results only show one red strip on the quality control line (C line), and no red strip on the detection line (T line); and if only the detection line has a red strip and the quality control line does not have a red strip, the detection result is invalid.
In the application method provided by the invention, the amplification reaction condition is constant temperature amplification at 40 ℃ for 10 min.
More specifically, the use method of the PDCoV nucleic acid detection test strip comprises the following steps:
(1) RNA extraction: extracting RNA of a sample to be detected by a conventional method;
(2) RT-RAA isothermal amplification: taking RNA extracted from a sample as a template, performing amplification reaction by using the RT-RAA kit of the PDCoV, and performing constant temperature amplification for 10min at 40 ℃;
(3) lateral flow strip (LFD) detection of amplification products: and detecting the amplification product by using the test strip, dripping the amplification product on a test strip sample pad, carrying out chromatography on the product along an NC membrane in the direction of absorbent paper, and forming a 'colloidal gold-labeled streptavidin-RNA amplification product' compound with two ends carrying Biotin and FAM labels respectively when an RNA amplification product to be detected exists, wherein the compound is intercepted by a mouse anti-FAM monoclonal antibody labeled at a T line on the NC membrane, and a red strip seen by naked eyes is formed after accumulation.
According to the understanding of the skilled person, the application of RT-RAA-LFD primer pairs, probes, kits and test strips for detecting PDCoV in the field of PDCoV detection and the application in visual judgment of PDCoV infection are also claimed.
The invention has the beneficial effects that:
the RT-RAA-LFD primer pair, the probe, the kit and the lateral flow test strip for detecting the PDCoV screen out the forward primer F3, the reverse primer mR7 and the probe mP which are suitable for quickly and visually detecting the PDCoV ORF1b gene on site aiming at the RT-RAA-LFD detection of the PDCoV, and can realize quick, specific, sensitive and simple detection of the PDCoV, thereby making up the defects of the prior detection technology.
The RT-RAA-LFD primer pair, the probe, the kit and the test strip of the PDCoV are used for carrying out amplification of a PDCoV target gene sequence based on the RT-RAA-LFD primer pair and the probe, and can be completed only by reacting for 10min at the constant temperature of 40 ℃.
Drawings
FIG. 1 is a schematic diagram of gene sequence information of a representative strain of 124 porcine delta-coronavirus and optimal primers and probes in the specific positions of ORF1b genes in the world, which are referred to by the invention; wherein the country or region of origin of the strain and its GenBank accession number are marked on the left side of the figure.
FIG. 2 is a schematic diagram of the distribution of 7 pairs of candidate primers and probes designed by the present invention for establishing the porcine delta-coronavirus RT-RAA-LFD in the most conserved region (16000-16213bp) of the diagnostic target ORF1b gene; wherein, F1-F7 represent 7 candidate upstream primers; R1-R7 represent 7 candidate downstream primers, and mR7 represents a primer after a one-nucleotide mutation (nucleotide 6, T → C) is introduced into R7; p represents an nfo probe, and mP represents a probe after two nucleotide mutations (nucleotide 4G → C, nucleotide 27A → G) have been introduced into the probe P.
FIG. 3 shows the result of 2% agarose gel electrophoresis of the RT-RAA primer screening provided by the present invention; wherein, (A) all 7 downstream primers R1, R2, R3, R4, R5, R6 and R7 are screened by using a randomly selected upstream primer F1; (B) screening all 7 upstream primers F1, F2, F3, F4, F5, F6 and F7 by using the screened optimal downstream primer R7; finally, F3 is proved to be the optimal upstream primer, and R6 is the suboptimal downstream primer (alternative); m: 8000bp marker.
FIG. 4 shows the RT-RAA-LFD detection results of two pairs of candidate primer pairs (F3/R7 and F3/R6) and probe (P) screened by the invention; wherein, 1: adding an upstream primer F3, a downstream primer R7, a probe P and 1.6X 10 to a reaction system5TCID50The result of RT-RAA-LFD amplification and detection is carried out on/mL PDCoV RNA; 2: adding an upstream primer F3, a downstream primer R7 and a probe P into a reaction system, and directly carrying out RT-RAA-LFD amplification and detection without adding PDCoV RNA; 3: adding an upstream primer F3, a downstream primer R6, a probe P and 1.6X 10 to a reaction system5TCID50The result of RT-RAA-LFD amplification and detection is carried out on/mL PDCoV RNA; 4: adding an upstream primer F3 and a downstream primer into a reaction systemR6, probe P, but not PDCoV RNA directly carries out RT-RAA-LFD amplification and detection; NTC represents no template control.
FIG. 5 shows the analysis of the base complementary pairing between the downstream primers (R7 and R6) and the probe (P) according to the present invention and its effect on the detection result of RT-RAA-LFD, wherein P represents the nfo probe without nucleotide mutation introduced; r6 and R7 represent downstream primers without nucleotide mutations introduced; mP represents the probe after two nucleotide mutations (nucleotide 4G → C, nucleotide 27A → G) have been introduced into probe P; mR7 represents the primer after introduction of a one-nucleotide mutation (nucleotide 6T → C) in R7; short lines between nucleotides: base complementary pairing possibly formed between the downstream primer and the probe; (B) in (1): adding an upstream primer F3, a downstream primer mR7, a probe mP and 1.6X 10 to a reaction system5TCID50The result of RT-RAA-LFD amplification and detection is carried out on/mL PDCoV RNA; 2: adding an upstream primer F3, a downstream primer mR7 and a probe mP into a reaction system, and directly carrying out RT-RAA-LFD amplification and detection without adding PDCoV RNA; NTC represents no template control.
FIG. 6 shows the optimal reaction temperature and reaction time for the RT-RAA-LFD according to the present invention; wherein, A: results of different reaction temperatures; b: analyzing optical density values of a quality control line (C line) and a detection line (T line) of the test strips with different reaction temperatures by using ImageJ software, and calculating and comparing a T/C ratio; c: results of different reaction times; d: and analyzing the optical density values of the C line and the T line of the test strip with different reaction times by using ImageJ software, and calculating and comparing the T/C ratio.
FIG. 7 shows the detection results of the RT-RAA-LFD specificity test according to the present invention; wherein, PCV 2: porcine circovirus type 2; PRV: porcine pseudorabies virus; and (3) PRRSV: porcine reproductive and respiratory syndrome virus; FMDV: foot and mouth disease virus; PEAV: porcine enteric coronavirus type a; PRoV: porcine rotavirus; TGEV: transmissible gastroenteritis virus of swine; PEDV: porcine epidemic diarrhea virus; NTC: no template control; PDCoV: porcine delta coronavirus; c: a quality control line; t: and detecting lines.
FIG. 8 shows the results of the RT-RAA-LFD sensitivity assay of the present invention; wherein (A) is PDCoV RT-RAA-LFD detectionThe result is; 1: 1.6X 107TCID50/mL PDCoV RNA；2：1.6×106TCID50/mL PDCoV RNA；3：1.6×105TCID50/mL PDCoV RNA；4：1.6×104TCID50/mL PDCoV RNA；5：1.6×103TCID50/mL PDCoV RNA；6：1.6×102TCID50/mL PDCoV RNA；7：1.6×101TCID50/mL PDCoV RNA；8：1.6×100TCID50/mL PDCoV RNA；9：1.6×10-1TCID50/mL PDCoV RNA；10：1.6×10-2TCID50/mL PDCoV RNA；11：1.6×10-3TCID50/mL PDCoV RNA; 12: no Template Control (NTC); c: a quality control line; t: detecting lines; (B) probability regression analysis of PDCoV RT-RAA-LFD detection result, wherein the detection limit of 95% confidence interval is 103.599TCID50/mL PDCoV RNA (equivalent to 3.97 TCID)50PDCoV RNA/reaction).
FIG. 9 shows the results of real-time fluorescent quantitative RT-PCR in comparison with RT-RAA-LFD in the present invention; wherein, (A) is PDCoV real-time fluorescence quantitative RT-PCR detection result; 1: 1.6X 107TCID50/mL PDCoV RNA；2：1.6×106TCID50/mL PDCoV RNA；3：1.6×105TCID50/mL PDCoV RNA；4：1.6×104TCID50/mL PDCoV RNA；5：1.6×103TCID50/mL PDCoV RNA；6：1.6×102TCID50/mL PDCoV RNA；7：1.6×101TCID50/mL PDCoV RNA；8：1.6×100TCID50/mL PDCoV RNA；9：1.6×10-1TCID50/mL PDCoV RNA；10：1.6×10-2TCID50/mL PDCoV RNA；11：1.6×10-3TCID50/mL PDCoV RNA; 12: no Template Control (NTC); (B) probability regression analysis of PDCoV real-time fluorescence quantitative RT-PCR, the detection limit of 95% confidence interval is 102.366TCID50/mL PDCoV RNA (equivalent to 0.232 TCID)50PDCoV RNA/reaction).
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 RT-RAA-LFD primer pairs and Probe design, screening and optimization of PDCoV
Downloading the whole genome sequences of 124 representative PDCoV strains at home and abroad in an NCBI database, comparing and analyzing the sequences by using MAFFT software (Version 7), selecting a region with the most conservative nucleotide sequence in the PDCoV genome and the length of about 213bp in ORF1b gene as a diagnostic target (as shown in figure 1), and respectively designing 7 upstream primers (F1-F7), 7 downstream primers (R1-R7) and 1 probe (P) according to the basic principle of RAA primer and probe design. All primer and probe sequence information is shown in Table 1, and their relative positions in ORF1b gene are shown in FIG. 2.
TABLE 1 primer pairs and probes for RT-RAA assay
To ensure the amplification efficiency of RT-RAA-LFD, it is necessary to screen 14 primers designed to cover the conserved region of ORF1b, so that a better primer and probe combination can be obtained. The primer screening strategy is as follows: randomly selecting a certain forward primer to screen all reverse primers, and then selecting the optimal reverse primer to screen all forward primers, thereby determining the optimal primer combination.
After amplifying an RNA template of a known amount of PDCoV CHN-HN-1601 strain (GenBank accession number MG832584.1) by RT-RAA, analyzing the amplification efficiency of different primers by combining 2% agarose gel electrophoresis, thereby screening out an optimal primer pair, and the main operation steps are as follows:
(1) preparation of RT-RAA reaction System
The RT-RAA reaction system (25.00. mu.L) comprises:
reaction buffer A14.70. mu.L;
reaction buffer B1.25. mu.L;
10 pmol/. mu.L of upstream primer 1.00. mu.L;
10 pmol/. mu.L downstream primer 1.00. mu.L;
1.00 mu L of PDCoV RNA template;
nuclease-free water 6.05. mu.L.
Negative control added nuclease-free water served as template, and positive control added RNA template of known PDCoV strains.
(2) Amplification of RT-RAA reaction systems
Setting reaction conditions: instantly centrifuging and mixing the reaction solution, and reacting in a constant temperature water bath kettle at 40 deg.C for 20 min;
and (3) analyzing an amplification product: taking 20 microliter of amplification product to carry out 2% agarose gel electrophoresis, and observing the result under ultraviolet rays;
quality control standard: if the negative control has no amplification band and the positive control has a specific amplification band, the test data is valid, otherwise, the test result is invalid;
and (3) describing and judging results: the sample to be detected has no amplification band, and the sample is judged to be negative; and a specific amplification band appears, and the sample is judged to be positive.
As shown in A of figure 3, F1 is selected as an upstream primer, the upstream primer is respectively matched with downstream primers R1-R7 for RT-RAA amplification, the brightness and abundance of bands of amplification products are analyzed through 2% agarose gel electrophoresis, R7 which generates the brightest band is determined as the optimal downstream primer, and the downstream primer R6 which has the band brightness second to R7 is used as an alternative downstream primer. Fixing the optimal downstream primer R7, then screening the 7 upstream primers F1-F7, and analyzing the band brightness and abundance of the amplified products by means of 2% agarose gel electrophoresis, as shown in B of figure 3, screening F3 as the optimal upstream primer.
F3 is selected to be respectively matched with R6 and R7 for RT-RAA-LFD detection, and an RT-RAA reaction system (25.00 mu L) comprises:
reaction buffer A14.70. mu.L;
reaction buffer B1.25. mu.L;
10 pmol/. mu.L of forward primer 1.00. mu.L;
10 pmol/. mu.L reverse primer 1.00. mu.L;
10 pmol/. mu.L probe 0.30. mu.L;
1.00 mu L of PDCoV RNA template;
nuclease-free water 5.75. mu.L.
Negative controls added nuclease-free water served as template, and other reactions added RNA template of known PDCoV strains. And (3) instantly centrifuging and uniformly mixing the reaction solution, placing the mixture in a constant-temperature water bath kettle at 40 ℃ for reacting for 20min, dropwise adding 50 mu L of 10-time diluted amplification product to a sample adding plate of a test strip, and reading the result after 1 min.
As shown in fig. 4, the optimal upstream primer F3 and the optimal downstream primer R7 are combined with the probe P to make the detection result positive or weakly positive to the PDCoV RNA template and the RT-RAA-LFD of the negative control (see test cards 1 and 2); and the optimal upstream primer F3 and the suboptimal downstream primer R6 are combined with the probe P, and the RT-RAA-LFD detection results of the PDCoV RNA template and the negative control are still positive (see test paper cards 3 and 4).
Under the premise of eliminating nucleic acid contamination, base complementary pairing possibly formed between reverse primers R7 and R6 and a probe P (namely, probe and Primer cross dimer can be formed) is analyzed by using Primer Premier 5.0 software, and according to the size of binding energy (delta G) for forming base complementary pairing between the Primer and the probe, a plurality of bases participating in forming dimer between a downstream Primer R7 and the probe P are finally determined to be mutated, so that the 4 th nucleotide G of the probe is mutated into C (G → C) and the 27 th nucleotide A is mutated into G (A → G), the 6 th nucleotide T of a downstream Primer R7 is mutated into C (T → C), and the capability of forming dimer is lost (A in figure 5). And (3) carrying out RT-RAA-LFD detection on the RNA templates of the negative control and the known PDCoV strain again by using the reverse primer mR7 and the probe mP after the base mutation, wherein as shown in B of figure 5, the RT-RAA-LFD detection results of the RNA templates of the negative control and the known PDCoV strain are respectively negative and positive, and the normal recovery is consistent with the expected result.
After the primer design, screening and modification, the optimal upstream primer for detecting the PDCoV nucleic acid RT-RAA-LFD is F3, the optimal downstream primer is mR7 and the optimal probe is mP.
In this example, the optimal primer combinations and probe sequences were finally screened as follows:
forward primer F3:
5’-AGTATAATGGCGTCCATCCAGCTCATGCTT-3’；
reverse primer mR 7:
5’-FAM-ACGTGTTAGGAACATATTATGATACGCATC-3’；
the 5' end of the reverse primer mR7 is modified by a fluorescent group FAM;
a probe mP:
5’-Biotin-GGTCCAGAGTACCGCTGTGAGGAGCCGCTTG[THF]TAAATTAGTAGGAGT-Phosphorylation-3’
the 5 'end of the probe mP is modified by biotin, the 32 nd base C is replaced by Tetrahydrofuran (THF), and the 3' end is modified by phosphorylation.
Example 2 optimization of optimal reaction temperature and time for RT-RAA-LFD detection of PDCoV
This example provides an optimization study of optimal reaction temperature and time for RT-RAA-LFD detection of PDCoV, comprising the following steps:
at 1.6X 105TCID50The optimum reaction temperature was determined by RT-RAA-LFD amplification using/mL PDCoV RNA as template and the optimized primer combination (F3/mR7) and probe (mP) and the above reaction system (25.00. mu.L) at 20, 25, 30, 35, 37, 40 and 42 ℃ for 20min, respectively. On the basis, the optimal reaction temperature, the same primer combination, probe and reaction system are adopted for 1.6 multiplied by 105TCID50the/mL PDCoV RNA templates were amplified for 0, 2, 5, 10, 15, 20, 25, and 30min, respectively, to determine the optimal reaction time.
As shown in A of FIG. 6, the detection results at 20 ℃ and 25 ℃ were negative, and the brightness of the detection line (T line) at 40 ℃ was significantly stronger than those at 30 ℃, 35 ℃ and 37 ℃ and comparable to that at 42 ℃; the light density values of a quality control line (C line) and a detection line of the test strip under different reaction temperature conditions are analyzed by means of ImageJ software, and the T/C ratio is calculated to further show that the brightness and the relative ratio of the detection line are highest at 40 ℃, so that 40 ℃ is selected as the optimal reaction temperature for detecting PDCoV by using RT-RAA-LFD. As shown in C of fig. 6, there are no or only blurred detection lines at 0min, 2min and 5min, while the detection lines at the other reaction times (10, 15, 20, 25 and 30min) have little difference in brightness; and analyzing the optical density values of the C line and the T line of the test strip under different reaction time conditions by means of ImageJ software, and further confirming that the brightness and the relative ratio of the detection line enter a plateau stage from 10min by calculating the T/C ratio, and selecting 10min as the optimal reaction time for detecting PDCoV by using RT-RAA-LFD in consideration of the timeliness of epidemic disease detection.
Example 3 specific assay for RT-RAA-LFD detection of PDCoV
The implementation carries out the specificity analysis of RT-RAA-LFD detection PDCoV, and the specific steps are as follows: the nucleic acids of the porcine delta coronavirus (PDCoV), the Porcine Epidemic Diarrhea Virus (PEDV), the porcine transmissible gastroenteritis virus (TGEV), the porcine rotavirus (PRoV), the porcine entero-A coronavirus (PEAV), the Foot and Mouth Disease Virus (FMDV), the Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), the porcine pseudorabies virus (PRV) and the porcine circovirus type 2 (PCV2) are respectively used as templates, the RT-RAA-LFD detection is carried out according to the method, and a template-free control (NTC) is arranged at the same time.
As shown in the results in FIG. 7, no bands were observed in the test lines (T-lines) corresponding to the PDCoV RNA templates, but in the test lines corresponding to the test strips. The result shows that the RT-RAA-LFD established by the invention can realize the specific detection of PDCoV and does not have cross reaction with other important porcine viruses.
Example 4 sensitivity assay for RT-RAA-LFD detection of PDCoV
This example provides a sensitivity assay for RT-RAA-LFD detection of PDCoV, the procedure is as follows:
PDCoV RNA diluted by 10 times of gradient is taken as a template (the titer of the corresponding PDCoV is 1.6 multiplied by 10)7TCID50/mL～1.6×10-3TCID50mL), nucleic acid amplification was performed under the optimal conditions for RT-RAA-LFD, and a no-template control (NTC) was set. Meanwhile, the real-time fluorescent quantitative RT-PCR method in the published literature (Pan Z., et al., Virulence,2020,11:707-718) is used for ordinary-time detection and comparison.
As shown in A of FIG. 8, when the concentration of the PDCoV RNA template is 1.6X 107TCID50/mL～1.6×103TCID50The detection result of RT-RAA-LFD is positive when the concentration is/mL, and the concentration of PDCoV RNA template is 1.6 multiplied by 102TCID50/mL～1.6×10-3TCID50At the time of/mL, the detection results of RT-RAA-LFD are all negative, which indicates that RT-RAA-LFD can detect 1.6 × 103TCID50/mL of PDCoV RNA. As shown in B of FIG. 8, by performing probabilistic regression analysis on the results of 8 independent RT-RAA-LFD detections, it was confirmed that when the confidence was set to 95%, the detection sensitivity of RT-RAA-LFD on PDCoV was 103.599TCID50/mL PDCoV RNA (equivalent to 3.97 TCID)50PDCoV RNA/reaction).
As shown in A of FIG. 9, when the concentration of the RNA template of PDCoV is 1.6X 107TCID50/mL～1.6×102TCID50When the concentration is/mL, the detection result of the real-time fluorescent quantitative RT-PCR is positive, and when the concentration of the PDCoV RNA template is 1.6 multiplied by 101TCID50/mL～1.6×10-3TCID50at/mL, the detection results of the real-time fluorescence quantitative RT-PCR are all negative, which indicates that the real-time fluorescence quantitative RT-PCR can detect 1.6 multiplied by 102TCID50/mL of PDCoV RNA. As shown in B of FIG. 9, by performing probabilistic regression analysis on the 8 independent real-time fluorescent quantitative RT-PCR detection results, it was confirmed that the detection sensitivity of the real-time fluorescent quantitative RT-PCR to PDCoV was 10 when the confidence was set to 95%2.366TCID50/mL PDCoV RNA (equivalent to 0.232 TCID)50PDCoV RNA/reaction).
Example 5 test application to actual clinical specimens
This example provides the testing of actual clinical samples by the following steps: 149 parts of clinical samples of pigs (comprising 23 parts of small intestine tissue of pigs, 81 parts of anal swab and 45 parts of serum) are taken, RNA of the samples is extracted according to the instruction of an RNA extraction reagent, and then RT-RAA-LFD detection is carried out on the RNA of all the samples according to the method, and meanwhile, 149 parts of clinical samples are detected and compared at ordinary times by utilizing a real-time fluorescent quantitative RT-PCR method in published literature (Pan Z., et al., Virulence,2020,11: 707-718).
As shown in Table 2, 149 clinical samples RT-RAA-LFD detected 71 positive, real-time fluorescence quantitative RT-PCR detected 75 positive, RT-RAA-LFD and real-time fluorescence quantitative RT-PCR detected the coincidence rate of 97.32% (145/149) for 149 clinical samples, Kappa mean value was 0.960, P < 0.001.
TABLE 2 clinical specimen test results
In conclusion, the RT-RAA-LFD method disclosed by the invention can realize rapid, specific, sensitive, visual and simple detection on the porcine delta coronavirus. The detection sensitivity of the method is equivalent to that of real-time fluorescent quantitative RT-PCR, but the detection speed of the method is obviously higher than that of the real-time fluorescent quantitative RT-PCR, and the method has simple requirements on instruments, does not need a temperature control instrument for thermal cycle reaction, and is suitable for the real-time visual detection of the infection of the porcine delta coronavirus on the basement layer or on the site.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (10)
1. The primer probe combination is characterized in that the sequences of the primers are shown as SEQ ID NO.3 and SEQ ID NO.16, and the sequence of the probe is shown as SEQ ID NO. 17.
2. The primer probe combination of claim 1, wherein the 5 'end of the probe is modified with biotin, the 32 nd base C is replaced with Tetrahydrofuran (THF), and the 3' end is modified with phosphorylation; modifying the 5' end of the reverse primer by using a fluorescent group FAM;
the probe sequence is as follows:
5’-Biotin-GGTCCAGAGTACCGCTGTGAGGAGCCGCTTG[THF]TAAATTAGTAGGAGT-Phosphorylation-3’
the reverse primer sequences are as follows:
5’-FAM-ACGTGTTAGGAACATATTATGATACGCATC-3’。
3. the primer probe combination of any one of claims 1-2, in the preparation of a PDCoV detection kit or a detection test strip.
4. An RT-RAA-LFD detection kit of PDCoV, which is characterized by comprising the primer probe combination of any one of claims 1 to 2.
5. The RT-RAA-LFD detection kit according to claim 4, wherein the RT-RAA-LFD detection kit further comprises a reaction buffer and enzymes.
6. The RT-RAA-LFD detection kit of claim 5, wherein the enzymes comprise reverse transcriptase, recombinase, DNA polymerase, single-stranded DNA binding protein, and endonuclease IV.
7. A PDCoV nucleic acid detection test strip, which is used for detecting the RT-RAA-LFD detection kit of any one of claims 4 to 6, wherein the product obtained by amplification comprises: a sample combination pad, a detection line and a quality control line; the sample combining pad contains colloidal gold labeled streptavidin, a detection line (T line) contains a mouse anti-FAM monoclonal antibody, and a quality control line (C line) contains biotin coupled with Bovine Serum Albumin (BSA).
8. The method of using the test strip of claim 7, comprising: amplifying RNA extracted from a sample to be detected by using the RT-RAA-LFD detection kit of any one of claims 4-6 by using the RNA as a template to obtain an amplification product, and performing visual interpretation on the amplification product by using the detection test strip;
the result judgment criteria are: two red strips appear in the positive result, one is positioned on a detection line (T line), and the other is positioned on a quality control line (C line); negative results only show one red strip on the quality control line (C line), and no red strip on the detection line (T line); and if only the detection line has a red strip and the quality control line does not have a red strip, the detection result is invalid.
9. The use method according to claim 8, wherein the reaction condition of the amplification is isothermal amplification at 40 ℃ for 10 min.
10. Use of the primer probe combination of any one of claims 1-2 or the RT-RAA-LFD detection kit of any one of claims 4-6 or the test strip of claim 7 for visual determination of PDCoV infection.
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