CN111518955A - HRM primer pair, kit and method for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus - Google Patents
HRM primer pair, kit and method for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus Download PDFInfo
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Abstract
The invention provides a HRM primer pair, a kit and a method for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus, and belongs to the technical field of molecular biology detection. The HRM primer pair is designed according to a sequence between 29000bp and 29500 bp of a full-length gene of the feline coronavirus FCOV. The FECV and FIPV can be identified and distinguished simultaneously through different shapes of melting curves by carrying out a reaction once, and technical support is provided for early diagnosis, prevention and control of diseases.
Description
Technical Field
The invention relates to an HRM primer pair, a kit and a method for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus, and belongs to the technical field of molecular biology detection.
Background
Feline coronavirus (FCoV) is an unfractionated, single-stranded positive-strand RNA virus belonging to the order Nidovirales (Nidovirales), the family Coronaviridae (Coronaviridae), and the genus Alphacoronavirus (Alphacoronavirus). FCoV has two biotypes, Feline Enteric Coronaviruses (FECV) and Feline Infectious Peritonitis Viruses (FIPV). Although most FCoV-infected cats suffer from mild or asymptomatic enteritis, 12% of cats will develop Feline Infectious Peritonitis (FIP), with a high fatality rate for FIP.
The existing experimental methods for detecting FECV and FCoV are mainly PCR methods, including common PCR and fluorescent quantitative PCR, and also indirect immunofluorescence and ELISA methods. The PCR technology has high detection sensitivity, wide application range, simple operation and other reasons. However, the PCR detection technology has complex procedures, needs precise instruments and has long detection time, thus being not beneficial to field detection in non-laboratory environment and popularization and application in basic laboratories.
A High-resolution melting curve (HRM) is a genetic typing method for gene mutation scanning and genotyping of different forms of melting curves formed based on different melting temperatures of mononucleotides, and the basic principle is that the thermal stability of double-stranded nucleotides (dsDNA) is influenced by the length and the base composition, the sequence change can cause the change of melting behavior of the dsDNA in the process of temperature rise, and the used saturated fluorescent dye can only be embedded and combined on the dsDNA. The kit has extremely high sensitivity, can detect the difference of single base without using a sequence specific probe, does not need post-treatment (such as enzyme digestion, electrophoresis and the like) on a PCR product, can realize real closed-tube operation so as to reduce the pollution risk, and has developed into an important means such as SNP genotyping, point mutation screening, species identification and the like.
Currently, there is no report on the identification of FECV and FIPV by HRM. Therefore, it is necessary to establish a HRM detection method for feline enterocoronavirus and feline infectious peritonitis virus.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an HRM primer pair, a kit and a method for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus.
The invention selects a specific gene sequence to design a pair of HRM primers, simultaneously identifies and distinguishes FECV and FIPV through different-shape melting curves, and provides technical support for early diagnosis, prevention and control of diseases.
In order to achieve the purpose, the invention adopts the technical scheme that:
an HRM primer pair for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus, wherein the sequences of an upstream primer and a downstream primer of the primer pair are respectively shown as SEQ ID No.1 and SEQ ID No. 2:
FECV-FIPV-F:5′-CTATGTGAAGTACTGTCCAC-3′(SEQ ID No.1)；
FECV-FIPV-R:5′-GTGAGGTCTGTCGAAGACCT-3′(SEQ ID No.2)。
the primer pair is designed according to a sequence between 29000bp and 29500 bp of the full-length gene of the feline coronavirus FCOV.
The invention also provides an HRM kit for rapidly identifying the feline enterocoronavirus and the feline infectious peritonitis virus, which comprises the primer pair.
The HRM kit also comprises other reagents required by HRM reaction: enzymes, fluorescent dyes, buffers, etc.;
the HRM kit also comprises a negative control and a positive control; the negative control is a water blank control without RNase; the positive control is a FECV positive sample and a FIPV positive sample, and the concentration is 1 mug/mL.
The reagent in the HRM kit can adopt a commercial reagent.
The invention also provides a method for identifying feline enterocoronavirus and feline infectious peritonitis virus, which comprises the following steps:
(1) extracting RNA of a sample to be detected as a template;
(2) carrying out RT-PCR amplification by using the primer pair of the invention;
(3) and HRM analysis is carried out according to the fluorescence signal, so that the feline enterocoronavirus and the feline infectious peritonitis virus can be rapidly identified.
The methods are useful for non-disease diagnostic and therapeutic uses.
The RT-PCR reaction system of step (2) totally contains 25. mu.l of RNA 2. mu.l, PrimerScript 1step Enzyme 1. mu.l, 2 × step Buffer 12.5. mu.l, RNase Free ddH2O5.5. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, fluorescent dye LCGREEN 2. mu.l.
The RT-PCR reaction program is 30min at 50 ℃ and 2min at 95 ℃. Pre-denaturation at 95 ℃ for 30 s; 10s at 95 ℃, 20s at 55 ℃, 30s at 72 ℃, 35 cycles, 5min at 72 ℃ and 30min at 4 ℃.
The step (3) HRM analysis: and (3) collecting fluorescence every 0.05s at the temperature rise rate of 0.1 ℃/step at 75-88 ℃ for carrying out dissolution curve analysis.
The sample is obtained from cat chest, abdominal dropsy, cerebrospinal fluid, aqueous humor, renal granuloma, cat nose, throat swab, feces, rectal swab, etc.
The invention also provides application of the HRM primer pair in preparation of products for detecting, identifying and distinguishing FECV and FIPV.
The inventor finds that the primer design on the conserved gene S or N commonly used by FCOV is not feasible through a large number of research experiments. Finally, HRM primers are designed on a relatively conserved sequence (the sequence between 29000bp and 29500 bp of the FCOV full-length gene sequence) on the 7b gene of the FCOV, the base difference exists between the FECV fragment amplified by the primers and the FIPV fragment, the peak value (Tm value) difference exists in a melting curve after the real-time fluorescence quantitative PCR reaction is finished, and the FECV and the FIPV can be distinguished by only using one primer through the HRM reaction.
The invention realizes that the FECV and the FIPV can be quickly and accurately identified and distinguished by only one pair of primers through HRM reaction for the first time. This is the greatest advantage of the present invention.
Drawings
FIG. 1 shows the sequence alignment results of FIPV and FECV fragment amplified by the primer pair HRM for identifying feline enterocoronavirus and feline infectious peritonitis virus according to the present invention.
FIG. 2 shows the result of HRM method specific assay for identifying feline enterocoronavirus and feline infectious peritonitis virus according to the present invention.
FIG. 3 shows the result of the sensitivity test of detecting FECV by HRM method for identifying feline enterocoronavirus and feline infectious peritonitis virus according to the present invention.
FIG. 4 shows the FIPV sensitivity test results of the HRM method for identifying feline enterocoronavirus and feline infectious peritonitis virus according to the present invention.
FIG. 5 shows the result of the HRM method for identifying feline enterocoronavirus and feline infectious peritonitis virus according to the present invention.
FIG. 6 shows the results of the HRM method for identifying feline enterocoronavirus and feline infectious peritonitis virus in clinical samples.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the present invention is not limited thereto. The methods of the present invention are those commonly used in the art unless otherwise specified, and the reagents of the present invention may be commercially available without further specification. The methods of the invention are useful for the diagnosis and treatment of non-diseases.
Example 1 design of HRM primers for the identification of feline Enterovirus and feline infectious peritonitis Virus
1.1 isolation and culture of Virus and nucleic acid extraction
FIPV was isolated and cultured by the laboratory and FECV nucleic acids were derived from the extraction of clinical samples. Nucleic acid RNA extraction was performed on ascites and intestinal fecal samples of cats according to the instructions of RNA extraction kit of TIANGEN, Inc.
1.2 design of HRM primers
Full length sequences of FECV and FIPV (Genebank accession numbers KY566209 and KC461237.1, respectively) were downloaded at NCBI for alignment. Attempts to design primers on commonly used conserved S or N genes have been unsuccessful. Finally, a pair of primers (the primer sequences are shown in Table 1) is designed on a relatively conserved sequence (between the 29000bp and 29500 bp sequence sites) on the 7b gene. The pair of primers can simultaneously carry out specific amplification on a 217bp homologous fragment of FECV and FIPV. The amplification results were identified by sequencing and compared by NCBI BLAST to determine that the amplified fragments belong to the same homologous sequence in FIPV and FECV (as shown in Table 2 and FIG. 1). Two subtypes of feline coronavirus were identified by differences in the Tm of the melting curves due to base differences. If a single specific peak appears at Tm (82.23 +/-0.05) DEG C, the FECV is judged to be positive; FIPV positivity was determined by the appearance of a single specific peak at Tm ═ 83.98. + -. 0.03 ℃ C.
TABLE 1 primer sequence Listing
| Primer numbering | Primer sequences | Number of bases | Amplified fragment length |
| FECV-FIPV-F | CTATGTGAAGTACTGTCCAC(SEQ ID No.1) | 20 | 87bp |
| FECV-FIPV-R | GTGAGGTCTGTCGAAGACCT(SEQ ID No.2) | 20 | 87bp |
TABLE 2
Example 2 establishment of HRM response for the identification of feline enteric coronavirus and feline infectious peritonitis Virus
The extracted nucleic acid was used as a template, and amplified using the upstream and downstream primers shown in Table 1. RT-PCR reactionThe reaction system consisted of 25. mu.l of 1. mu.l of RNA, 1. mu.l of PrimerScript 1step Enzyme, 12.5. mu.l of 2 × step Buffer, RNase Free ddH2O5.5. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, fluorescent dye LC GREEN 1. mu.l. The working concentration of the upstream primer and the downstream primer is 10 mu mol/L.
The RT-PCR reaction program is 50 ℃ 30min 95 ℃ 2 min. Pre-denaturation at 95 ℃ for 30 s; 10s at 95 ℃, 20s at 55 ℃, 30s at 72 ℃, 35 cycles, 5min at 72 ℃ and 30min at 4 ℃. In the HRM analysis, the dissolution curve analysis was performed by collecting fluorescence every 0.05s at a temperature rise rate of 0.1 ℃/step at 75 ℃ to 88 ℃.
The result shows that the HRM method for simultaneously identifying the feline enterocoronavirus and the feline infectious peritonitis virus through one-time reaction is successfully established.
Example 3 HRM method specificity assay for the identification of feline Enterovirus and feline infectious peritonitis Virus of the invention
To analyze whether the HRM reaction has detection specificity among other canine/feline pathogens on the basis of being able to distinguish FECV from FIPV, the HRM reaction was performed using nucleic acids of the following samples as templates: canine Coronavirus (CCV), Canine Parvovirus (CPV), Canine Distemper (CDV), Canine Adenovirus (CAV), Feline Herpesvirus (FHV), Feline Parvovirus (FPV), Feline Calicivirus (FCV), 1 negative control (ddH)20) One FECV positive sample, one FIPV positive sample.
The results showed that the samples were negative except for FECV and FIPV (see FIG. 2). The method is proved to have good specificity and no cross reaction with other pathogens.
Example 4 HRM method sensitivity assay for the identification of feline enteric coronavirus and feline infectious peritonitis Virus of the invention
Positive plasmid standards for FECV and FIPV were constructed, respectively. Each positive plasmid standard was diluted 10-fold and HRM reaction was performed with the diluted sample as a template. The template concentration of the plasmid standards for FECV and FIPV was 10 from high to low8copies/μL～101copies/. mu.L, 8 gradients total. The results showed that the lower detection limits of FECV and FIPV were 102copies/uL (shown in FIGS. 3 and 4, respectively).
Example 5 HRM method repeatability test for identifying feline Enterovirus and feline infectious peritonitis Virus according to the invention
Based on the difference of the melting curve specificity peak Tm value, FECV and FIPV are differentially diagnosed. If a single specific peak appears at Tm (82.23 +/-0.05) DEG C, the FECV is judged to be positive; if a single specific peak appears at Tm (83.98 +/-0.03) DEG C, the FIPV is judged to be positive; the standard deviation is between 0.01 and 1, the fluctuation is small, and the repeatability is good. The test results are shown in FIG. 5, which shows that the method of the present invention has good repeatability.
Example 6 HRM method for identifying feline Enterovirus and feline infectious peritonitis Virus according to the invention for assay of clinical samples
HRM detection was performed on 21 clinical samples using the constructed HRM detection method, and the results showed that 7 of them were positive for FECV, the Tm average was 82.05 ℃, and the positive rate was 33%. 2 parts of FIPV positive, the Tm average value is 83.77 ℃, and the positive rate is 9.52%. The results are shown in FIG. 6, and the results measured by the method of the present invention are consistent with the actual 100%.
Example 7 kit for identifying feline Enterovirus and feline infectious peritonitis Virus
The kit comprises: the sequence is shown as SEQ ID No.1, the sequence is shown as SEQ ID No.2, the sequence is shown as downstream primer, Enzyme, Buffer, RNase Free ddH2O, fluorescent dye, negative control, positive control, etc.
SEQUENCE LISTING
<110> Guangdong province laboratory animal monitoring station
<120> HRM primer pair, kit and method for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus
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<170>PatentIn version 3.3
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agttataagg caacccgatg tttaaaactg gtctttccga ggaattactg gtcatcgcgc 60
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Claims (10)
1. An HRM primer pair for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus is characterized in that sequences of an upstream primer and a downstream primer of the primer pair are respectively shown as SEQ ID No.1 and SEQ ID No. 2.
2. The HRM primer pair of claim 1, wherein the primer pair is designed based on the sequence of the FCOV full-length gene from 29000bp to 29500 bp.
3. An HRM kit for rapidly identifying feline enterocoronavirus and feline infectious peritonitis virus, wherein the kit comprises the primer pair of claim 1.
4. The HRM kit of claim 3, further comprising enzymes, fluorescent dyes and other reagents required for HRM reaction.
5. The HRM kit of claim 4, further comprising a negative control and a positive control; the negative control is a water blank control without RNase; the positive control is a FECV positive sample and a FIPV positive sample.
6. A method for identifying feline enterocoronavirus and feline infectious peritonitis virus, comprising the steps of:
(1) extracting RNA of a sample to be detected as a template;
(2) performing RT-PCR amplification with the primer pair of claim 1;
(3) performing HRM analysis according to the fluorescence signal to quickly identify the feline enterocoronavirus and the feline infectious peritonitis virus;
the methods are useful for non-disease diagnostic and therapeutic uses.
7. The method according to claim 6, wherein the RT-PCR reaction system of step (2) is 25. mu.l of RNA 2. mu.l, PrimerScript 1step Enzyme 1. mu.l, 2 × step Buffer 12.5. mu.l, RNase Free ddH2O5.5. mu.l, upstream primer 1. mu.l, downstream primer 1. mu.l, fluorescent dye LC GREEN 2. mu.l.
8. The method as set forth in claim 6, wherein the RT-PCR reaction procedure of step (2) is 50 ℃ for 30min, 95 ℃ for 2min, 95 ℃ for 30s, 95 ℃ for 10s, 55 ℃ for 20s, 72 ℃ for 30s, 35 cycles, 72 ℃ for 5min, and 4 ℃ for 30 min.
9. The method as claimed in claim 6, wherein the step (3) of HRM analysis: and (3) collecting fluorescence every 0.05s at the temperature rise rate of 0.1 ℃/step at 75-88 ℃ for carrying out dissolution curve analysis.
10. Use of the HRM primer pair of claim 1 in the preparation of a medicament for detecting and discriminating FECV and FIPV.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626272A (en) * | 2020-12-25 | 2021-04-09 | 中国医学科学院病原生物学研究所 | Novel coronavirus SARS-CoV-2 detection and molecular typing method and kit |
| CN114835804A (en) * | 2022-05-19 | 2022-08-02 | 安徽中起生物科技有限公司 | Feline infectious peritonitis egg yolk antibody composition and preparation method and application thereof |
| CN116042538A (en) * | 2022-11-24 | 2023-05-02 | 华中农业大学 | Cat coronavirus strain and application thereof |
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| CN102791886A (en) * | 2010-01-18 | 2012-11-21 | 乌得勒支大学控股有限公司 | Means and methods for distinguishing FECV and FIPV |
| CN108359743A (en) * | 2017-12-27 | 2018-08-03 | 中国农业科学院特产研究所 | A kind of HRM primer group for differentiating FPV and CPV, the kit containing the primer sets and its application |
| CN110714098A (en) * | 2019-11-20 | 2020-01-21 | 上海市动物疫病预防控制中心(上海市兽药饲料检测所、上海市畜牧技术推广中心) | Composition and kit for detecting feline calicivirus and feline infectious peritonitis virus and application of composition and kit |
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| CN116042538A (en) * | 2022-11-24 | 2023-05-02 | 华中农业大学 | Cat coronavirus strain and application thereof |
| CN116042538B (en) * | 2022-11-24 | 2024-05-07 | 华中农业大学 | Cat coronavirus strain and application thereof |
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