A fluorescent quantitative PCR detection kit for avian leukosis virus based on pol gene and application thereof

Abstract

The application discloses an avian leukosis virus fluorescent quantitative PCR detection kit based on a pol gene and application thereof. The application is based on a fluorescent quantitative PCR technology, and specific primers and a TaqMan probe aiming at a pol gene conservative region of avian leukosis virus are designed to realize specific amplification and detection of virus nucleic acid. The core improvement of the application is that after nucleic acid is extracted from animal tissue, plasma and other samples, a DNA enzyme is added to perform digestion treatment, and residual host genome DNA in the sample is specifically degraded, so that a false positive problem caused by amplification of an endogenous pol gene is completely eliminated, and the specificity and accuracy of detection are obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection, specifically relating to a quantitative real-time PCR detection kit for avian leukosis virus based on the pol gene and its application. Background Technology

[0002] Avian leukosis (AL) is a neoplastic disease caused by avian leukosis virus (ALV), resulting in significant economic losses for the poultry industry. ALV has multiple subgroups (such as A, B, J, and K subgroups), with substantial differences in gene sequences between them, posing a challenge to universal detection of ALV. In practice, based on the characteristics of ALV retroviruses, the entire viral genome is inserted into the host genome. Typically, after nucleic acid extraction, primers are designed based on the ALV env gene sequence for direct PCR or qPCR. However, there is currently no universal method for the detection of avian leukosis virus using quantitative real-time PCR, especially when selecting the env gene as the target gene. Due to its high variability, it often leads to false negatives and missed detections. Furthermore, endogenous avian leukosis virus (eALV) gene fragments are widely present in the genome of birds (especially chickens), forming part of the host genome and existing in DNA form. If total DNA is extracted directly for PCR, false positives are very likely to occur. When detecting exogenous infections, it is crucial to avoid interference from these endogenous fragments.

[0003] Although the pol gene is relatively conserved across different ALV subgroups and is an ideal target for developing universal detection methods, the pol gene sequence of endogenous retroviruses also exists in the avian host genome, which can easily lead to non-specific amplification, produce false positive results, and affect the accuracy of the detection results.

[0004] Therefore, establishing a highly sensitive ALV detection method that can accurately distinguish between exogenous ALV and host genomic background is crucial for the eradication and control of avian leukosis. Existing detection methods include virus isolation and ELISA, but these methods have drawbacks such as low sensitivity, which makes ALV eradication difficult. Quantitative real-time PCR (qPCR) technology has advantages such as high sensitivity, high specificity, rapid and efficient operation, and quantification, and has been widely used in the detection of pathogenic microorganisms. Summary of the Invention

[0005] Objective of this invention: The objective of this invention is to provide a simple, rapid, highly sensitive, and highly specific quantitative real-time PCR detection kit for avian leukosis virus (ALV) based on the pol gene. This invention leverages the genetic characteristics of ALV and improves the accuracy and reliability of detection by effectively eliminating residual ALV DNA genes in the host genome.

[0006] Another objective of this invention is to provide the application of the aforementioned detection kit, which effectively solves the core problem of host genomic DNA interference in the prior art through a key DNase digestion step, thereby enabling accurate and specific quantitative detection of the pol gene transcription level of avian leukosis virus.

[0007] Technical solution: To solve the above-mentioned technical problems, the present invention provides a real-time PCR detection kit for avian leukosis virus based on the pol gene. The detection kit includes specific primer pairs as shown in SEQ ID NO.1 and SEQ ID NO.2 and a probe as shown in SEQ ID NO.3; the kit also includes DNase.

[0008] The detection kit also includes a reverse transcription reagent.

[0009] The test kit also includes a positive control and a negative control.

[0010] The positive control is a plasmid containing the plasmid shown in SEQ ID NO.4.

[0011] The application of the detection kit described in this invention in the in vitro quantitative detection of the pol gene.

[0012] This invention also provides a method for in vitro quantitative detection of the pol gene, the detection method comprising the following steps:

[0013] (1) Collect poultry samples and extract total nucleic acid from the samples;

[0014] (2) The total nucleic acid obtained in step (1) was digested using DNase;

[0015] (3) The total RNA after step (1) was reverse transcribed into cDNA using a one-step method. The cDNA was then subjected to qPCR reaction. The Ct value of the sample was obtained by using a real-time PCR instrument. The content of the pol gene was obtained by comparing it with the standard curve.

[0016] The sample mentioned in step (1) includes one or more of the following: poultry blood plasma, tissue sample, meconium, egg white or cell culture sample.

[0017] The DNase treatment conditions in step (2) are as follows: 7.5 U of DNase is added to every 10 μL of reaction system, and digestion is carried out at 37°C for 15 minutes.

[0018] In step (2), the one-step reverse transcription fluorescence quantitative PCR reaction uses a commercially available premixed solution to integrate reverse transcription and PCR amplification into a single reaction.

[0019] The qPCR reaction in step (3) includes the specific primer pairs shown in SEQ ID NO.1 and SEQ ID NO.2 and the probe shown in SEQ ID NO.3.

[0020] This invention, based on quantitative real-time PCR technology, utilizes designed specific primers and TaqMan probes targeting the conserved region of the avian leukosis virus (avian leukosis virus) pol gene to achieve specific amplification and detection of viral nucleic acid. The core improvement of this invention lies in the following: after extracting nucleic acid from animal tissues, plasma, and other samples, DNase is added for digestion treatment, specifically degrading residual host genomic DNA in the samples. This completely eliminates false positives caused by endogenous pol gene amplification, significantly improving the specificity and accuracy of detection.

[0021] Beneficial effects: Compared with existing detection technologies, the present invention has the following significant advantages,

[0022] 1. This invention fundamentally solves the problem of false positives caused by host genomic DNA interference by introducing DNase into the detection kit and digesting the sample with DNase before detection, thus achieving extremely high specificity.

[0023] 2. This invention utilizes real-time PCR technology combined with optimized primers and probes to significantly improve detection sensitivity. The detection kit of this invention can detect viral RNA as low as 10 copies / reaction.

[0024] 3. The detection method of the present invention is fast and efficient. It adopts a one-step cDNA synthesis technology, which completes reverse transcription in one reaction. The entire detection process can be completed within 2.5 hours.

[0025] 4. This invention is easy to operate, easy to standardize, has simple steps, good repeatability, and is suitable for promotion in routine testing laboratories. Attached Figure Description

[0026] Figure 1 Real-time quantitative PCR amplification curves of standards with serial dilutions from 10^1 to 10^7 copies / μL;

[0027] Figure 2The standard curve plot shows the linear relationship between the gradient dilution of the standard from 10^1 to 10^7 copies / μL and the Ct value, with R² > 0.99;

[0028] Figure 3 This is a comparison chart of the effects of DNase treatment, showing that the SPF sample without treatment has an amplification curve, while the treated sample has no amplification curve.

[0029] Figure 4 The specificity verification diagram shows that the method of the present invention amplifies ALV-J, A, and B positive samples, but does not amplify other avian pathogens and SPF samples. Detailed Implementation

[0030] Experimental materials for this invention:

[0031] Positive plasma for avian leukosis virus (ALV-J, ALV-A, etc.) and negative plasma for SPF chickens (preserved in this laboratory).

[0032] Viral DNA / RNA Extraction Kit (Tianlong Biotechnology Co., Ltd.) (Catalog No.: T014)

[0033] DNA enzyme (Aikerui Biotechnology) (Catalog No.: AG12001)

[0034] One-step reverse transcription real-time PCR kit (Yifeixue Biotechnology) (Catalog No.: YFXM0009)

[0035] Pro Taq HS Premixed Probe-Based qPCR Kit (Akerui Biotechnology) (Catalog No.: AG11704)

[0036] Real-time quantitative PCR instrument (Roche) (Model LightCycler 96)

[0037] Example 1

[0038] (1) Sample collection:

[0039] Eleven plasma samples were collected from SPF chickens artificially infected with ALV-J, and nine plasma samples were collected as negative controls.

[0040] (2) Nucleic acid extraction and DNA digestion:

[0041] Total RNA was extracted from plasma samples using a commercial nucleic acid extraction kit (Tianlong Biotechnology Co., Ltd., catalog number: T014). After RNA extraction, DNase (Aikerui Biotechnology, catalog number: AG12001) was added according to the kit instructions. 7.5 U of DNase was added to a 10 μL reaction system and digested at 37°C for 15 minutes.

[0042] (3) Primer and probe design:

[0043] Specific primers and TaqMan probes were designed targeting the conserved regions of the avian leukosis virus (ALV) pol gene. Validation via NCBIL alignment showed that the following sequences exhibit high specificity for major ALV subgroups, including ALV-J, A, B, and K:

[0044] Forward primer ALV-pol-F: 5'-atatctatgcagcaggctagggagg-3'

[0045] Reverse primer ALV-pol-R: 5'-ccatatctgtaggggtcccaaac-3'

[0046] Probe ALV-pol-P: 5'-[CY5]-gcaggggctgaattacaatgcgg-[BHQ1]-3'

[0047] (4) Reverse transcription and quantitative PCR reaction:

[0048] The reaction was performed using a one-step reverse transcription kit (Yifexue Biotechnology, catalog number: YFXM0009). The reaction system was as follows: 4×gDNA Removal Mix: 5 μL, 4× Supreme-Enzyme Mix: 5 μL; RNA template: 2 μL; RNase-free Water: to a final volume of 20 μL. The reverse transcription reaction program was: gDNA removal: 37℃, 5 minutes; reverse transcription: 55℃, 15 minutes. The qPCR reaction system was as follows (using 20 μL as an example): 2×Mix: 10 μL, forward primer (10 μM): 0.5 μL, reverse primer (10 μM): 0.5 μL, probe (10 μM): 0.5 μL, RNA template: 2 μL, RNase-free Water: to a final volume of 20 μL. PCR reaction program: 37℃: 120s, 95℃: 120s; PCR cycle (40 cycles): denaturation: 95℃, 10s; annealing / extension: 60℃, 30s (CY5 channel fluorescence signal is acquired at this step).

[0049] Simultaneously, the ALV-J pol gene fragment (GenBank accession number GU982308) was synthesized and ligated into a T vector to obtain a plasmid containing the ALV-J pol gene fragment. This plasmid was used as a standard plasmid and serially diluted 10-fold (10^7 to 10^1 copies / μL). Its Ct value and real-time fluorescence intensity were measured using the same PCR amplification program to establish a standard curve.

[0050] (5) Results Analysis:

[0051] The Ct value for each sample was obtained using the analysis software of the real-time PCR instrument. See the results below. Figure 1 A positive result was defined as a Ct value less than 36 and the appearance of a typical S-shaped amplification curve; a Ct value greater than or equal to 36 or no Ct value was considered negative. A standard curve was constructed using plasmid standards with known copy numbers, and the viral RNA in the samples was absolutely quantified using the standard curve. The results of the standard curves are shown in Table 1 and [Table data missing]. Figure 2 .

[0052] Table 1 Standard Curve and Sensitivity Measurement

[0053]

[0054] Standard curve parameters: slope = -3.293, intercept = 39.37, correlation coefficient R² = 0.9998, amplification efficiency E = 100.9%.

[0055] The method of the present invention exhibits excellent linearity in the linear range (10^1 to 10^7 copies / μL), with a detection limit as low as 10 copies / μL and extremely high sensitivity.

[0056] The fluorescence quantitative PCR method of the present invention was compared with the traditional virus isolation method, ELISA method, and ordinary RT-PCR + electrophoresis. The results are shown in Table 2.

[0057] Table 2 Comparison of the method of the present invention with traditional methods

[0058]

[0059] Example 2: Effect of DNAse treatment on detection specificity

[0060] This embodiment is basically the same as the method in Embodiment 1. The difference is that the sample types include ALV-J positive plasma #1, SPF negative plasma #1, and SPF negative plasma #2. The difference also lies in whether the samples are treated with DNase or not. The detection results are shown in Table 3.

[0061] Table 3. Effect of DNAse treatment on detection specificity

[0062]

[0063] Table 2 clearly shows that for positive samples, the virus can be detected regardless of whether DNase treatment is performed (the difference in Ct values ​​may be due to a slight influence of DNA background on amplification efficiency). However, for virus-free SPF negative serum, untreated samples all showed significant false-positive signals (Ct values ​​between 29 and 33), while treated samples all yielded negative results. This fully demonstrates the necessity and crucial role of the DNase digestion step in eliminating host genomic DNA interference and ensuring the specificity of test results.

[0064] Example 3 Repeatability Experiment

[0065] This embodiment is basically the same as the method in Embodiment 1. The difference is that experiments were conducted to verify the method under different concentrations of standard plasmid samples (10^5 copies / μL, 10^3 copies / μL, and 10^1 copies / μL).

[0066] Table 4 Repeatability Test Results

[0067]

[0068] The method of this invention exhibits good repeatability and stability, with intra-batch and inter-batch coefficients of variation both less than 5% at different concentration levels.

[0069] Example 4: Specificity Experiment of Chicken Meconium

[0070] This embodiment is basically the same as the method in Embodiment 1, except that the samples were obtained from SPF chicken negative anal swab 1, SPF chicken negative anal swab 2, SPF chicken negative anal swab 3, SPF chicken negative anal swab 4, SPF chicken negative anal swab 5, SPF chicken positive anal swab 6, Marek's disease virus MDV (infecting CEF cells), chicken infectious anemia virus CAV (infecting MSB1 cells), and avian reticuloendothelial proliferative virus REV (infecting DF-1 cells) preserved in the laboratory. Total nucleic acid was extracted using a nucleic acid extraction instrument and reverse transcription was performed using the Yifeixue one-step method kit. The results are shown in Table 5.

[0071] Table 5 Meconium and Specificity

[0072]

[0073] Conclusion: As can be seen from Table 5, the method of this invention has high specificity and cross-reactivity with other pathogens, and can detect ALV in cloacal anal test samples.

[0074] Example 5: Clinical Sample Application

[0075] This embodiment uses the same method as Embodiment 1, except that the clinical samples are derived from cell cultures. The clinical sample detection method of this embodiment is compared with the conventional ELISA method, and the results are shown in Table 6.

[0076] Table 6 Comparison of results of clinical sample cell cultures detected by the method of this invention and ELISA method

[0077]

[0078] Conclusion: It can be seen that the qRT-PCR method can detect all ELISA-positive samples, and also detect positive samples that ELISA cannot detect.

Claims

1. A real-time PCR detection kit for avian leukosis virus based on the pol gene, characterized in that, The detection kit includes specific primer pairs as shown in SEQ ID NO.1 and SEQ ID NO.2 and a probe as shown in SEQ ID NO.3; the kit also includes a DNA enzyme.

2. The detection kit according to claim 1, characterized in that, The test kit also includes a reverse transcription reagent.

3. The detection kit according to claim 1, characterized in that, The test kit also includes a positive control and a negative control.

4. The detection kit according to claim 3, characterized in that, The positive control is a plasmid containing the plasmid shown in SEQ ID NO.

4.

5. The application of the detection kit according to any one of claims 1 to 4 in the in vitro quantitative detection of the pol gene.

6. A method for in vitro quantitative detection of the pol gene, characterized in that, The detection method includes the following steps: (1) Collect poultry samples and extract total nucleic acid from the samples; (2) The total nucleic acid obtained in step (1) was digested using DNase; (3) The total RNA after step (1) was reverse transcribed into cDNA using a one-step method. The cDNA was then subjected to qPCR reaction. The Ct value of the sample was obtained by using a real-time PCR instrument. The content of the pol gene was obtained by comparing it with the standard curve.

7. The method for in vitro quantitative detection of the pol gene according to claim 6, characterized in that, The sample mentioned in step (1) includes one or more of the following: avian plasma, tissue sample, meconium, egg white or cell culture sample.

8. The method for in vitro quantitative detection of the pol gene according to claim 6, characterized in that, The DNase treatment conditions described in step (2) are as follows: 7.5 U of DNase is added to every 10 μL of reaction system, and digestion is carried out at 37°C for 15 minutes.

9. The method for in vitro quantitative detection of the pol gene according to claim 6, characterized in that, The one-step reverse transcription fluorescence quantitative PCR reaction described in step (2) uses a commercially available premixed solution to integrate reverse transcription and PCR amplification into a single reaction.

10. The method for in vitro quantitative detection of the pol gene according to claim 6, characterized in that, The qPCR reaction described in step (3) includes the specific primer pairs shown in SEQ ID NO.1 and SEQ ID NO.2 and the probe shown in SEQ ID NO.3.

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