CN117385100A - Fluorescence quantitative PCR detection kit for canine respiratory coronavirus - Google Patents

Abstract

The invention discloses a fluorescence quantitative PCR detection kit for canine respiratory coronavirus, and relates to the technical field of molecular biology detection. The fluorescent quantitative PCR detection kit comprises a primer pair with a nucleotide sequence shown as SEQ ID NO.3-4 and a probe with a nucleotide sequence shown as SEQ ID NO. 5. The detection kit for the canine respiratory coronavirus provided by the invention detects the canine respiratory coronavirus by adopting a fluorescent quantitative PCR method, and has the advantages of strong specificity, good repeatability and high sensitivity for canine respiratory coronavirus detection, and the lowest detection concentration can reach 3.3X10 ¹ The copies/. Mu.L provides good technical support for diagnosis of the canine respiratory disease.

Description

Fluorescence quantitative PCR detection kit for canine respiratory coronavirus

Technical Field

The invention relates to the technical field of molecular biology detection, in particular to a fluorescent quantitative PCR detection kit for canine respiratory coronavirus.

Background

Coronavirus is an enveloped virus with a large (27-32 kb) single stranded positive sense RNA genome. Structural proteins located within the viral envelope are spike protein (S), membrane protein (M), envelope protein (E) and Hemagglutinin Esterase (HE) proteins, the latter only being present in group 2 members of the coronavirus. Nucleocapsid protein (N) binds to viral RNA to form a helical nucleocapsid. Canine respiratory coronavirus (CRCoV), unlike the well-known enteritis canine coronavirus, belongs to the genus beta coronavirus, is one of the causative agents of Canine Infectious Respiratory Disease (CIRD), is commonly found in the early stages of canine infectious respiratory disease, mainly invading the upper respiratory tract and causing mild respiratory symptoms, and is clinically susceptible to mixed infection with other respiratory pathogens. The nucleotide homology of CRCoV spike gene and bovine coronavirus (BCoV) spike gene is 97.3%, and the nucleotide homology of CRCoV spike gene and human coronavirus OC43 (HCoV-OC 43) is 96.9%. The virus is most commonly present in the trachea and pharyngeal tonsils, and the current commonly used diagnostic kit adopts an RT-PCR method, but the method has lower sensitivity, can not completely detect positive samples with low virus content, is easy to miss, and is not beneficial to disease diagnosis. Therefore, it is necessary to develop a new detection kit to improve the sensitivity and specificity of detection, so as to achieve no missing detection and no error detection for sample detection.

Disclosure of Invention

The invention aims to provide a fluorescence quantitative PCR detection kit for the canine respiratory coronavirus, which solves the problems in the prior art, has strong specificity, high sensitivity and good repeatability for canine respiratory coronavirus detection, and provides good technical support for canine respiratory disease diagnosis.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a primer pair and probe combination for detecting canine respiratory coronavirus, which comprises a primer pair with nucleotide sequences shown as SEQ ID NO.3-4 and a probe with nucleotide sequences shown as SEQ ID NO. 5.

Further, the 5 'end of the probe is marked with FAM group, and the 3' end is marked with MGB group.

The invention also provides application of the primer pair and the probe combination in preparing a fluorescence quantitative PCR detection kit for the canine respiratory coronavirus.

The invention also provides a fluorescence quantitative PCR detection kit for the canine respiratory coronavirus, which comprises the primer pair and the probe combination.

Further, the fluorescent quantitative PCR detection kit also comprises Taq Pro HS Universal U +probe Master Mix and RNase-free ddH ₂ O。

Further, the reaction system for performing fluorescent quantitative PCR detection by the fluorescent quantitative PCR detection kit is a 10 mu L reaction system and comprises Taqpro HS Mix, an upstream primer, a downstream primer, a probe, a template and RNase-free ddH ₂ O；

The concentration of the upstream primer and the downstream primer in the reaction system is 0.8 mu mol/L; the concentration of the probe in the reaction system was 0.4. Mu. Mol/L.

Further, the reaction procedure of the fluorescent quantitative PCR detection kit for fluorescent quantitative PCR detection comprises: 120s at 37 ℃;95 ℃ for 30s;95℃for 5s,58℃for 30s,40 cycles.

The invention discloses the following technical effects:

the detection kit for the canine respiratory coronavirus provided by the invention detects the canine respiratory coronavirus by adopting a fluorescence quantitative PCR method, and has the advantages of strong specificity, good repeatability, high sensitivity and concentrated lowest detection for the canine respiratory coronavirus detectionThe degree can reach 3.3X10 ¹ copies/μL。

The fluorescent quantitative PCR of the invention is a reaction system of 10 mu L, compared with a conventional reaction system of 20 mu L, the cost is saved, and the sensitivity is basically indistinguishable from the conventional reaction system of 20 mu L.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the running results of the M gene amplification product of example 1;

FIG. 2 is a result of ordinary PCR verification by the fluorescent quantitative PCR primer of example 2;

FIG. 3 is the result of the reaction system optimization experiment in example 2;

FIG. 4 is the result of the reaction procedure optimization experiment in example 2;

FIG. 5 is a fluorescence quantitative PCR amplification curve of different concentration standard plasmids in example 2; 1 to 7 are respectively 3.3X10 ¹ -3.3×10 ⁷ copies/μL；

FIG. 6 is a fluorescent quantitative PCR standard curve established in example 2;

FIG. 7 shows the sensitivity test results of fluorescent quantitative PCR in example 3; 1 to 5 are respectively 3.3X10 ⁵ -3.3×10 ¹ copies/μL；

FIG. 8 is the result of a sensitivity test of the general PCR in example 3; 1 to 7 are respectively 3.3X10 ⁷ -3.3×10 ¹ cobies/. Mu.L; 8 is a negative control;

FIG. 9 is the result of the specificity experiment in example 4;

FIG. 10 is the result of the repeatability experiment in example 5; 1 to 5 are respectively 3.3X10 ¹ -3.3×10 ⁵ copies/μL；

FIG. 11 is a fluorescence quantitative PCR amplification curve of the different reaction systems of example 7.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

EXAMPLE 1 construction of Standard plasmid

The relatively conserved M gene was selected to construct a standard plasmid, and the M gene was first amplified, with the amplification primer sequences shown in Table 1.

Extracting CRCoV genome RNA by adopting a conventional DNA extraction method, carrying out reverse transcription, and carrying out PCR reaction by taking the obtained cDNA as a template, wherein a PCR reaction system and a reaction program are as follows:

every 20 mu L of the reaction system of the PCR reaction is: 10. Mu.M of upstream primer 1. Mu.L, 10. Mu.M of downstream primer 1. Mu.L, taq Mix 10. Mu.L, genomic cDNA 2. Mu.L, ddH ₂ O 6μL。

The PCR reaction procedure was: pre-denaturation at 95℃for 3min, denaturation at 95℃for 15s, annealing at 56℃for 15s, extension at 72℃for 40s,35 cycles, and extension at 72℃for 5min.

After the PCR reaction is finished, the amplified product is subjected to agarose gel electrophoresis with the voltage of 120V and the electrophoresis time of 28min, and according to the amplified band size judgment result, when the amplified band is 693bp, the M gene sequence of CRCoV is obtained.

TABLE 1M Gene amplification primers

The gel recovery was performed by conventional methods and the gel running results are shown in fig. 1.

The amplified fragment was ligated to the PCAGGS vector (length 4756 bp) using a conventional method to give a standard plasmid (length 5449 bp) which was sequenced correctly by the Optimago. The standard plasmid concentration was 20 ng/. Mu.L, and the copy number of the standard plasmid mother liquor was calculated to be 3.3X10 ⁹ copies/μL。

Example 2

1. Design of primer and probe

Selecting the most conserved segment design probe in M genes, and carrying out primer screening according to a few pairs of probe design, wherein the finally determined primer probe sequences are shown in Table 2:

TABLE 2 fluorescent quantitative PCR primer and probe sequences

Full length: 103bp

The fluorescent quantitative PCR primer is subjected to common PCR verification, and the gel running chart is shown in figure 2.

2. Reaction system and reaction program for optimizing fluorescent quantitative PCR method

2.1 laboratory apparatus and materials

The instrument used was an analytikjena (yerba germany) qPCR instrument, and the probe enzyme used was Taq Pro HS Universal U + probe Master Mix from Vazymeg corporation, hereinafter omitted as tappro HS Mix.

In order to increase the sensitivity of the reaction, the reaction system, i.e., the use concentrations of the probe and the primer, is first determined. The probe is synthesized by the biological synthesis of the Optimus and the ingredients are dry powder. Using RNase-free ddH ₂ O was diluted to 100. Mu. Mol/L as a mother liquor. The primer is synthesized by the biological reaction of the family of the engine, and the concentration is 10 mu mol/L.

Using RNase-free ddH ₂ The probes were diluted to 2. Mu. Mol/L, 3. Mu. Mol/L, 4. Mu. Mol/L, 5. Mu. Mol/L, 6. Mu. Mol/L, and 7. Mu. Mol/L, respectively, by O.

Using RNase-free ddH ₂ The primers were diluted to 4. Mu. Mol/L, 6. Mu. Mol/L, 8. Mu. Mol/L, 10. Mu. Mol/L by O.

2.2 optimization of the reaction System

qPCR was performed using CRCoV cDNA as a template, using the primers and probes shown in Table 2, according to the reaction procedures shown in Table 4, and according to the reaction systems shown in Table 3:

TABLE 3 reaction System for screening experiments

TABLE 4 reaction procedure used in screening experiments

The reaction results are shown in Table 5 and FIG. 3, wherein the red color is thickened to a probe concentration of 4. Mu. Mol/L and the primer concentration is 8. Mu. Mol/L. The curve Ct value is smaller, the sensitivity is higher, and the amplification efficiency is higher. Therefore, the primers and probes at this concentration were selected as the final system, and the final optimized reaction system is shown in Table 6.

TABLE 5

TABLE 6 optimized reaction System

2.3 optimization of the reaction procedure

The experiment mainly determines the annealing temperature, which has a large influence on the extension.

Using the CRCoV cDNA as a template and the primers and probes shown in Table 2, a fluorescent quantitative PCR reaction was performed with the reaction system shown in Table 6, and with the annealing temperatures set at 52, 54, 56, 58 and 60℃on the basis of Table 4, respectively. The experiment results are shown in FIG. 4, with three replicates for each temperature. The results showed that the amplification efficiency and sensitivity were highest at 58℃and therefore this temperature was selected as the final annealing temperature, i.e., the reaction procedure after final optimization, as shown in Table 7:

TABLE 7 optimized reaction procedure

3. Establishing a standard curve

Using RNase-free ddH ₂ O Standard plasmid was diluted to 3.3X10 ¹ -3.3×10 ⁷ The fluorescence quantitative PCR was performed using primers and probes shown in Table 2, the reaction system was shown in Table 6, and the reaction procedure was shown in Table 7. Amplification results are shown in FIG. 5A standard curve between Ct values and log standard concentration is established from the amplification results (fig. 6), standard curve equation: y= -3.53x+38.66, r ² =0.999. As can be seen from the standard curve equation, the different concentration gradients are linearly related to the Ct value, and R ² And the correlation is good when the correlation is larger than 0.99.

As can be seen from the amplification curve, the exponential growth phase curve is parallel, reflects that the amplification efficiency of PCR is similar, the Ct values between different dilutions are uniform, and the Ct values and the copy number show good linear relationship.

Example 3 sensitivity experiment

1. Fluorescent quantitative PCR

Using RNase-free ddH ₂ O Standard plasmid was diluted to 3.3X10 ¹ -3.3×10 ⁵ The fluorescence quantitative PCR was performed using primers and probes shown in Table 2, the reaction system was shown in Table 6, and the reaction procedure was shown in Table 7. The amplification results are shown in FIG. 7. When the Ct value is less than 35, the result is judged as positive, and when the Ct value is greater than or equal to 35, the result is judged as negative.

2. Ordinary PCR

Using RNase-free ddH ₂ O Standard plasmid was diluted to 3.3X10 ¹ -3.3×10 ⁷ The probes/. Mu.L was subjected to a conventional PCR assay (conventional CRCoVPCR assay), the primer sequences used are shown in Table 8, and the reaction system and reaction procedure are as follows:

every 20 mu L of the reaction system of the PCR reaction is: 10. Mu.M of upstream primer 1. Mu.L, 10. Mu.M of downstream primer 1. Mu.L, 2 XTaq Mix 10. Mu.L, genome cDNA 2. Mu.L, ddH ₂ O 6μL。

The PCR reaction procedure was 95℃pre-denaturation for 3min,95℃denaturation for 15s,56℃annealing for 15s,72℃extension for 15s,35 cycles, and finally 72℃extension for 5min.

TABLE 8 primers for general PCR

FIG. 8 shows a gel chart of the results of the general PCR.

Fluorescent light meterThe detection results of quantitative PCR and general PCR showed that: the minimum detection concentration of the fluorescent quantitative PCR is 3.3X10 ¹ The lowest detection concentration of common PCR was 3.3X10. Mu.L of copies/ ⁴ The sensitivity of fluorescent quantitative PCR was about 1000-fold higher than that of ordinary PCR.

Example 4 specificity experiments

Specific experiments were performed by using CAV1 (canine adenovirus type I), CAV2 (canine adenovirus type II), CIV (canine influenza virus), CCV (canine enterovirus), CPIV (canine parainfluenza virus), CDV (canine distemper virus) and CPV (canine parvovirus), and the reaction system and reaction procedure are shown in Table 6 and Table 7, respectively.

All viral genomes are extracted by conventional genomic DNA or RNA extraction methods, and RNA viruses are reverse transcribed by conventional methods.

The experimental results are shown in FIG. 9, and the results show that only CRCoV has specific amplification and other pathogens have no amplification, so that the detection method has good specificity.

Example 5 repeatability experiments

Using RNase-free ddH ₂ O Standard plasmid was diluted to 3.3X10 ¹ -3.3×10 ⁵ The fluorescence quantitative PCR was performed using primers and probes shown in Table 2, the reaction system was shown in Table 6, and the reaction procedure was shown in Table 7. The experimental results are shown in fig. 10 and table 9, and the results show that the variation coefficient of the repetition between groups and the repetition in the groups is less than 2%, and the repeatability is good.

Table 9 results of repeatability experiments

Example 6 clinical sample detection

Samples of different areas are collected for detection. All samples were submitted for genome and reverse transcription using conventional methods. The sample was subjected to both ordinary PCR and fluorescent quantitative PCR assays, both assays being the same as in example 3.

The experimental results are shown in Table 10, and the results show that the detection rate of the fluorescent quantitative PCR established by the invention is obviously higher than that of the current common PCR for the detection of clinical samples.

TABLE 10 clinical sample test results

10.10. Mu.L System compared to 20. Mu.L System

Using RNase-free ddH ₂ O Standard plasmid was diluted to 3.3X10 ⁵ The fluorescence quantitative PCR was performed using the primers and probes shown in Table 2 in an amount of 10. Mu.L (shown in Table 6) and 20. Mu.L (double the amount of all components used on the basis of Table 6 while maintaining the concentrations of the components) respectively, and the reaction procedures were as shown in Table 7.

As shown in FIG. 11, the results showed that the sensitivity was substantially indistinguishable from that of the 20. Mu.L system, and only the amplification efficiency was slightly different, and the results were substantially unaffected.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims (7)

1. A primer pair and probe combination for detecting canine respiratory coronavirus is characterized by comprising a primer pair with a nucleotide sequence shown as SEQ ID NO.3-4 and a probe with a nucleotide sequence shown as SEQ ID NO. 5.

2. The primer pair and probe combination of claim 1, wherein the probe is labeled with a FAM group at the 5 'end and an MGB group at the 3' end.

3. Use of a primer pair and probe combination according to claim 1 or 2 for the preparation of a fluorescent quantitative PCR detection kit for canine respiratory coronavirus.

4. A kit for fluorescent quantitative PCR detection of canine respiratory coronavirus, comprising the primer pair and probe combination of claim 1 or 2.

5. The kit for fluorescent quantitative PCR according to claim 4, further comprising Taq Pro HS Universal U + probe Master Mix and RNase-free ddH ₂ O。

6. The kit for fluorescent quantitative PCR detection according to claim 5, wherein the reaction system for fluorescent quantitative PCR detection is 10. Mu.L reaction system comprising Taq pro HS Mix, upstream primer, downstream primer, probe, template and RNase-free ddH ₂ O；

The concentration of the upstream primer and the downstream primer in the reaction system is 0.8 mu mol/L; the concentration of the probe in the reaction system was 0.4. Mu. Mol/L.

7. The fluorescent quantitative PCR detection kit of claim 5, wherein the reaction procedure for performing fluorescent quantitative PCR detection with the fluorescent quantitative PCR detection kit comprises: 120s at 37 ℃;95 ℃ for 30s;95℃for 5s,58℃for 30s,40 cycles.