A pathogen multiplex PCR rapid detection method based on GenomeLabTM GeXP multiplex gene expression analysis technology
By using GenomeLab™ GeXP multiplex gene expression analysis technology, combined with two-step PCR amplification and capillary electrophoresis analysis using specific primers and fluorescently labeled universal primers, the problems of long time consumption and low efficiency of traditional methods have been solved, enabling rapid and accurate detection of multiple swine pathogens and supporting efficient disease monitoring.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SICHUAN ANIMAL SCI ACAD
- Filing Date
- 2026-01-19
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies are insufficient for rapid, accurate, and automated detection of various highly pathogenic pathogens in pig farms. Traditional methods are time-consuming, costly, inefficient, and lack consistent results and sensitivity.
Using GenomeLab™ GeXP multiplex gene expression analysis technology, specific primer combinations and fluorescently labeled universal primers were designed. Combined with two-step PCR amplification and capillary electrophoresis analysis, simultaneous detection of classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine parvovirus, porcine circovirus type 3, Fusobacterium necrosis, and porcine epidemic diarrhea virus was achieved.
It achieves high-throughput and accurate pathogen detection within 4 hours, with high specificity and sensitivity, avoiding false negatives and false positives, and providing objective and accurate results to support early warning and efficient monitoring of diseases in pig farms.
Smart Images
Figure CN122105006A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of pathogen detection technology, specifically to a rapid multiplex PCR detection method for pathogens based on GenomeLab™ GeXP multiplex gene expression analysis technology. Background Technology
[0002] In the process of large-scale and intensive development of pig farming, the outbreaks and epidemics of highly pathogenic pathogens such as classical swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine parvovirus, porcine circovirus type 3, clostridium necrophorum, and porcine epidemic diarrhea virus have become major threats to the healthy development of the industry. Rapid diagnosis is crucial for epidemic control.
[0003] However, existing detection technologies have significant shortcomings, making it difficult to meet the urgent needs of the current aquaculture industry for rapid and accurate disease monitoring. Traditional methods of pathogen isolation, culture, and biochemical identification are cumbersome and time-consuming, typically requiring 4 to 7 days, delaying the optimal time for epidemic control. Serological tests are susceptible to cross-reaction interference, resulting in limited specificity and sensitivity. Although polymerase chain reaction (PCR) technology, especially real-time quantitative PCR, has been widely used due to its high sensitivity and specificity, its throughput is limited. A single reaction can usually only detect one or a few pathogens, making it costly and inefficient when facing mixed infections of multiple pathogens or large-scale disease screening. Ordinary multiplex PCR technology attempts to improve efficiency by detecting multiple targets in a single reaction, but faces severe challenges in primer design. Different primers are prone to dimerization or competitive inhibition, leading to severely uneven amplification efficiency, weak or even complete loss of amplification signals for some targets, poor consistency and repeatability of results, and difficulty in guaranteeing reliability. In addition, conventional PCR products are mostly analyzed by agarose gel electrophoresis. This method has low resolution, makes it difficult to distinguish nucleic acid fragments of similar length, and is cumbersome to operate, easily introduces human interpretation errors, and is difficult to automate and standardize.
[0004] Therefore, there is an urgent need to establish a detection method that can simultaneously, rapidly, and with high throughput detect multiple highly lethal pathogens in pig farms, and possesses high specificity, high sensitivity, and high automation, in order to overcome many shortcomings of existing technologies and provide key technical support for the effective prevention and control of swine diseases. Summary of the Invention
[0005] Technical problems to be solved To address the problems mentioned in the background art, this invention provides a rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology.
[0006] Technical solution To achieve the above objectives, the present invention provides the following technical solution: A rapid multiplex PCR detection method for pathogens based on GenomeLab™ GeXP multiplex gene expression analysis technology uses a primer set consisting of 7 pairs of specific primers, 1 pair of positive control primers and 1 pair of universal primers to detect pathogens; Among them, the seven pairs of specific primers specifically bind to the target genes of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine parvovirus (PPV), porcine circovirus type 3 (PCV3), clostridium necrophorum (Fn), and porcine epidemic diarrhea virus (PEDV). The 5' end of the universal upstream primer is fluorescently labeled.
[0007] Furthermore, the nucleotide sequences of the specific primers are as follows: Classical Swine Fever Virus (CSFV) specific primer sequences are shown in SEQ ID NO:1 and SEQ ID NO:2; Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) specific primer sequences are shown in SEQ ID NO:3 and SEQ ID NO:4; Pseudorabies Virus (PRV) specific primer sequences are shown in SEQ ID NO:5 and SEQ ID NO:6; Porcine Parvovirus (PPV) specific primer sequences are shown in SEQ ID NO:7 and SEQ ID NO:8; Porcine Circovirus Type 3 (PCV3) specific primer sequences are shown in SEQ ID NO:13 and SEQ ID NO:14; Fusobacterium necrophorum (Fn) specific primer sequences are shown in SEQ ID NO:9 and SEQ ID NO:10; and Porcine Epidemic Diarrhea Virus (PEDV) specific primer sequences are shown in SEQ ID NO:11 and SEQ ID NO:12.
[0008] Furthermore, the nucleotide sequences of the positive control primers are shown in SEQ ID NO:15 and SEQ ID NO:16.
[0009] Furthermore, the nucleotide sequences of the universal primers are shown in SEQ ID NO:17 and SEQ ID NO:18.
[0010] Furthermore, the fluorescent label is a Cy5 fluorescent label.
[0011] Furthermore, the rapid detection method specifically includes the following steps: a) extracting nucleic acid from the sample to be tested and preparing a template; b) performing multiplex PCR amplification using the template; c) performing capillary electrophoresis analysis on the PCR amplification products; d) determining the presence or absence of the pathogen based on the fluorescence signal peak of a specific length fragment in the capillary electrophoresis pattern.
[0012] Furthermore, the multiplex PCR amplification procedure described in step b) includes: a first stage: performing 10-20 cycles at a higher annealing temperature for specific amplification using the specific primers and positive control primers; and a second stage: performing 10-20 cycles at a lower annealing temperature for universal amplification using the universal primers.
[0013] Furthermore, the annealing temperature in the first stage is 65°C, and the number of cycles is 15; the annealing temperature in the second stage is 50°C, and the number of cycles is 15.
[0014] Furthermore, the multiplex PCR reaction system described in step b) comprises: 0.1-0.3 μL each of the specific primer and the positive control primer at a concentration of 0.1 mM, 0.5-1.5 μL each of the universal primer at a concentration of 1.0 mM, 3-7 μL of 5×PCR buffer, 0.5-1.5 μL of 10 mM dNTPs, 1-3 μL of 5 U / μL DNA polymerase, 0.5-1.5 μL of the template, and sterile deionized water.
[0015] Further, the multiplex PCR reaction system in step b) is 25 μL, comprising: 0.2 μL each of the specific primer and positive control primer at a concentration of 0.1 mM, 1.0 μL each of the universal primer at a concentration of 1.0 mM, 5.0 μL of 5×PCR buffer, 1.0 μL of 10 mM dNTPs, 2.0 μL of 5 U / μL DNA polymerase, 1.0 μL of the template, and sterile deionized water.
[0016] Furthermore, the criteria for determination in step d) are as follows: if a positive control peak of 217bp appears, and product peaks with signal values greater than 50,000 appear at 135bp, 161bp, 181bp, 228bp, 268bp, 312bp, or 344bp, then it is determined to be positive for classical swine fever virus, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, clostridium necrophorum, pseudorabies virus, porcine parvovirus, or porcine circovirus type 3, respectively.
[0017] Beneficial technical effects This invention utilizes a combination of carefully designed specific chimeric primers and fluorescently labeled universal primers, along with a two-step PCR amplification procedure, to successfully achieve rapid and accurate detection of seven important pathogens—swine fever virus, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine parvovirus, porcine circovirus type 3, Fusobacterium necrosis, and porcine epidemic diarrhea virus—in a single reaction system. It boasts high throughput, enabling batch sample analysis within 4 hours, significantly improving detection efficiency. The method exhibits excellent specificity, showing no cross-reactivity with non-target pathogens, and effectively avoids false negatives and false positives through a built-in positive control, ensuring the reliability of the results. Furthermore, the method possesses high sensitivity, with a detection limit of 10² copies / μL for the target pathogen, meeting the clinical requirements for detecting low-load pathogens. Finally, automated fluorescent fragment analysis based on capillary electrophoresis ensures objective and accurate result interpretation, overcoming the subjectivity and low resolution limitations of traditional gel electrophoresis. This provides a powerful technical tool for high-throughput, automated monitoring and early warning of diseases in pig farms. Attached Figure Description
[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figures 1-3 These are the specificity test results of the GeXP multiplex PCR method of this invention.
[0020] Figures 4-6 The results show the sensitivity test results of the GeXP multiplex PCR method of this invention. Detailed Implementation
[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0022] The instruments and reagents used in the embodiments of this invention are from the following sources: The GeXP multiplex gene expression analysis system, formamide solution, separation buffer, and separating gel were all purchased from Beckman Coulter, Inc., USA.
[0023] The gradient PCR instrument was purchased from Cole-Parmer, USA.
[0024] The benchtop high-speed refrigerated centrifuge and the small benchtop high-speed centrifuge were purchased from Eppendorf, Germany.
[0025] The RNA / DNA Extraction Kit, plasmid pMD19-T, PUCM-T vector SpeⅠ restriction endonuclease, and T7 in vitro transcription kit were all purchased from Bestbio Biotechnology Co., Ltd.; the initial transcription reagent was purchased from TaKaRa; the Gel Extraction Kit and Plasmid Mini Kit were both purchased from Omega; and the GeXP Quick Start Kit was purchased from Beckman Coulter.
[0026] Example 1
[0027] This embodiment provides a GeXP multiplex PCR primer set for detecting seven common and highly lethal pathogens in pig farms. The primer set includes seven pairs of specific primers, one pair of positive control primers, and one pair of universal primers. The seven pathogens, their target genes, and the lengths of the amplified fragments are as follows: Classical swine fever virus targets the E2 gene, with a fragment length of 135 bp.
[0028] The target gene of porcine epidemic diarrhea virus is the N protein gene, with a fragment length of 161 bp.
[0029] The target gene of porcine reproductive and respiratory syndrome virus is the ORF5 gene, with a fragment length of 181 bp.
[0030] The target gene of *Clostridium necrophorum* is the FimA gene, with a fragment length of 228 bp.
[0031] The pseudorabies virus targets the GP4 gene, with a fragment length of 268 bp.
[0032] Porcine parvovirus targets the NS1 gene, with a fragment length of 312 bp.
[0033] Porcine circovirus type 3, with the target gene being the ORF2 gene, has a fragment length of 344 bp.
[0034] The positive control target gene was the pUCm-T plasmid with a fragment length of 217 bp.
[0035] The nucleotide sequences of each primer are shown in Table 1 below: Table 1
[0036] Example 2
[0037] This embodiment provides a rapid detection method for pathogens using multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology. The primer set composed in Example 1 is used to detect the pathogen. The specific GeXP multiplex PCR reaction system is as follows: 0.2 μL each of the 0.1 mM specific primers (SEQ ID NO:1-NO:16), 1.0 μL each of the 0.1 mM universal primers (SEQ ID NO:17-NO:18), 5.0 μL of 5×PCR Buffer, 1.0 μL of 10 mM dNTP mixture, 2.0 μL of 5 U / μL hot-start DNA polymerase, 1.0 μL of cDNA / DNA template to be tested, 0.2 μL of 50 ng / μL pUCm-T positive control plasmid, and sterile deionized water to a final volume of 25 μL. The annealing procedure is as follows: pre-denaturation at 95℃ for 2 minutes; then 95℃ for 40 seconds, 65℃ for 40 seconds, 72℃ for 30 seconds, for 15 cycles; 95℃ for 40 seconds, 50℃ for 40 seconds, 72℃ for 30 seconds, for 15 cycles; finally, extend at 72℃ for 5 minutes.
[0038] The specificity and sensitivity of the method in Example 2 will now be tested.
[0039] Specificity test: Positive samples containing classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine parvovirus (PPV), porcine circovirus type 3 (PCV3), *Fusobacterium necrophorum* (Fn), and porcine epidemic diarrhea virus (PEDV) were used as the target pathogen group. Positive samples containing enterotoxigenic *Escherichia coli* (ETEC), *Salmonella*, *Staphylococcus aureus*, and *Haemophilus parasuis* (HPS) were used as the non-target pathogen group. Sterile deionized water was used as the negative control. Nucleic acid was extracted from all the above samples. Amplification was performed strictly according to the GeXP multiplex PCR reaction system and annealing procedure established in Example 2. After amplification, 8 μL of ddH2O was mixed with 2 μL of PCR product, and the mixture was gently mixed to prepare 10 μL of diluted product, thus obtaining the diluted PCR product. Subsequently, a 40 μL loading system was prepared according to the following formula: 38.5 μL formamide, 0.5 μL DNA molecular weight standard (MARKER 400), and 1.0 μL diluted PCR product. The loading system was vortexed to mix thoroughly, followed by a brief, rapid centrifugation to remove droplets from the tube wall and air bubbles. The mixed solution was added to the sample wells of the GeXP system's dedicated loading plate, and then a drop of mineral oil was carefully added to each well to cover the liquid surface and prevent evaporation. 3 / 4 volume of separation buffer was added to each well of the corresponding buffer plate. The loading plate and buffer plate were then correctly placed in the slot of the GeXP multiplex PCR system, and the system's preset fragment analysis program was run for high-resolution capillary electrophoresis. After electrophoresis, the instrument's multiplex PCR system software automatically generated the electrophoresis pattern. The specificity of the GeXP multiplex PCR method for detecting the seven target pathogens was determined by the positive control peak in the electrophoresis pattern. The test results are as follows: Figures 1-6 As shown.
[0040] Depend on Figures 1-3 The results show that Figure 1 As a negative control, Figure 1 The presence of only a single, tall peak at 217 nt indicates that the PCR reaction system itself functioned normally and was free from contamination. However, apart from the positive control, no target pathogens were detected. The results were as expected. Figure 2 This is a fully positive control. Figure 2 The presence of a clear main peak demonstrates that this detection method can simultaneously and specifically detect all seven target pathogens in a single reaction, with each peak well separated and without significant interference. Figure 3 For specificity verification results, Figure 3 The only clear and significant main peak is still located at 217 nt, indicating that the primer set of this invention did not specifically bind to the genome of non-target pathogens, thus proving the high specificity of the detection method.
[0041] Sensitivity test: Using PCV3-positive plasmids with known genome copy numbers as templates, viral samples were serially diluted 10-fold to prepare a concentration of 10... 2 , 10 1 , 10 0 Template solution was prepared at a concentration of 1 copy / μL. Each dilution of template was used for GeXP multiplex PCR detection according to the method in Example 2, with each concentration tested three times. After amplification, capillary electrophoresis analysis was performed, and the lowest template concentration at which a specific fluorescence peak stably appeared at 344 bp was determined as the detection limit of this method. The test results are as follows: Figures 4-6 As shown.
[0042] Depend on Figures 4-6 The results show that the detection sensitivity of the method of the present invention is 10. 2 Copy / μL (detection rate 100%). In some cases, the sensitivity can even reach 10. 1 The LOD is defined as 10 copies / μL to ensure the stability and reliability of the results. 2 The number of copies / μL indicates that this method has extremely high sensitivity and can effectively address situations with low pathogen loads in clinical samples, providing reliable technical support for early diagnosis and warning.
[0043] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.
[0044] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.
[0045] Those skilled in the art should understand that the above descriptions are merely several specific embodiments of the present invention, and not all embodiments.
Claims
1. A rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology, characterized in that, The pathogen was detected using a primer set consisting of 7 pairs of specific primers, 1 pair of positive control primers, and 1 pair of universal primers. The 7 pairs of specific primers specifically bind to the target genes of classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies virus (PRV), porcine parvovirus (PPV), porcine circovirus type 3 (PCV3), Fusobacterium necrosis Fn, and porcine epidemic diarrhea virus (PEDV). The 5' end of the universal upstream primer is fluorescently labeled.
2. The rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology as described in claim 1, characterized in that, The nucleotide sequences of the specific primers are as follows: Classical Swine Fever Virus (CSFV) specific primer sequences are shown in SEQ ID NO:1 and SEQ ID NO:2; Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) specific primer sequences are shown in SEQ ID NO:3 and SEQ ID NO:4; Pseudorabies Virus (PRV) specific primer sequences are shown in SEQ ID NO:5 and SEQ ID NO:6; Porcine Parvovirus (PPV) specific primer sequences are shown in SEQ ID NO:7 and SEQ ID NO:8; Porcine Circovirus Type 3 (PCV3) specific primer sequences are shown in SEQ ID NO:13 and SEQ ID NO:14; Fusobacterium necrophorum (Fn) specific primer sequences are shown in SEQ ID NO:9 and SEQ ID NO:10; and Porcine Epidemic Diarrhea Virus (PEDV) specific primer sequences are shown in SEQ ID NO:11 and SEQ ID NO:
12.
3. The rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology as described in claim 1, characterized in that, The nucleotide sequences of the positive control primers are shown in SEQ ID NO:15 and SEQ ID NO:
16.
4. The rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology as described in claim 1, characterized in that, The nucleotide sequences of the universal primers are shown in SEQ ID NO:17 and SEQ ID NO:
18.
5. The rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology according to claim 1, characterized in that, The fluorescent label is a Cy5 fluorescent label.
6. The rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology according to claim 1, characterized in that, Specifically, the steps include: a) extracting nucleic acid from the sample to be tested and preparing a template; b) performing multiplex PCR amplification using the template; c) performing capillary electrophoresis analysis on the PCR amplification products; and d) determining the presence or absence of the pathogen based on the fluorescence signal peaks of specific length fragments in the capillary electrophoresis pattern.
7. The rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology according to claim 6, characterized in that, The multiplex PCR amplification procedure described in step b) includes: a first stage: performing 10-20 cycles at a higher annealing temperature for specific amplification using the specific primers and positive control primers; and a second stage: performing 10-20 cycles at a lower annealing temperature for universal amplification using the universal primers.
8. The rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology according to claim 7, characterized in that, The annealing temperature in the first stage is 65°C, and the number of cycles is 15; the annealing temperature in the second stage is 50°C, and the number of cycles is 15.
9. A rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology according to claim 6, characterized in that, The multiplex PCR reaction system described in step b) comprises: 0.1-0.3 μL each of the specific primer and the positive control primer at a concentration of 0.1 mM, 0.5-1.5 μL each of the universal primer at a concentration of 1.0 mM, 3-7 μL of 5×PCR buffer, 0.5-1.5 μL of 10 mM dNTPs, 1-3 μL of 5 U / μL DNA polymerase, 0.5-1.5 μL of the template, and sterile deionized water.
10. A rapid detection method for pathogen multiplex PCR based on GenomeLab™ GeXP multiplex gene expression analysis technology according to claim 6, characterized in that, The criteria for determination in step d) are as follows: if a positive control peak of 217bp appears, and product peaks with signal values greater than 50,000 appear at 135bp, 161bp, 181bp, 228bp, 268bp, 312bp, or 344bp, then the test is determined to be positive for classical swine fever virus, porcine epidemic diarrhea virus, porcine reproductive and respiratory syndrome virus, clostridium necrophorum, pseudorabies virus, porcine parvovirus, or porcine circovirus type 3, respectively.