Combination of primers, kit and method for double detection of haemophilus parasuis and porcine reproductive and respiratory syndrome virus and application
By designing specific primer combinations for Haemophilus parasuis and porcine reproductive and respiratory syndrome virus, and combining them with RPA and CRISPR-Cas systems, efficient and specific simultaneous detection under isothermal conditions was achieved. This solves the problems of insufficient specificity and low sensitivity in the existing technology for simultaneous identification, and is suitable for rapid detection and field applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHENYANG AGRI UNIV
- Filing Date
- 2026-05-08
- Publication Date
- 2026-06-09
AI Technical Summary
The existing technology lacks primer combinations that can simultaneously and specifically amplify Haemophilus parasuis and porcine reproductive and respiratory syndrome virus in the same reaction system. This results in insufficient specificity and low sensitivity when the two pathogen targets are identified simultaneously in the isothermal amplification system, which cannot meet the needs of rapid and accurate detection of mixed infections.
Specific primer combinations were designed targeting the Haemophilus parasuis 16S rDNA gene and the porcine reproductive and respiratory syndrome virus ORF7 gene. Combined with recombinase polymerase amplification (RPA) and CRISPR-Cas system, synchronous amplification and fluorescence signal detection under isothermal conditions were achieved. The components and reaction conditions of the dual-enzyme system were optimized to ensure matching amplification efficiency and specificity.
It achieves efficient and specific simultaneous detection of two pathogens in mixed infection samples under constant temperature conditions, with a sensitivity of 3.0×10-8 ng/μL. It is suitable for on-site point-of-care testing and can be adapted for pig farm screening, port quarantine and epidemiological monitoring.
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Figure CN122168783A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of molecular diagnostic technology for animal diseases, specifically involving primer combinations, reagent kits, methods, and applications for the dual detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus. Background Technology
[0002] Haemophilus parasuis ( Glaesserella parasuis , G. parasuis Porcine germ cell disease (PGD) causes Glaser's disease, characterized by serositis, arthritis, and pneumonia. Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive disorders in sows and respiratory syndrome in piglets. The two often co-infect or become secondary infections, leading to more complex and severe diseases in pigs and causing significant economic losses.
[0003] Currently used for detection G. parasuis Methods for detecting PRRSV include bacterial culture, PCR-related techniques, and immunological methods. Bacterial culture, considered the "gold standard," is time-consuming (24-72 hours), making it unsuitable for rapid diagnosis. PCR technology is accurate and highly sensitive, but relies on thermal cycling equipment, is complex and costly, and requires trained professionals, making it unsuitable for grassroots or field testing. Immunological methods, while simple and rapid, often lack sufficient sensitivity and specificity. More importantly, all of these methods are designed for single pathogens, which is insufficient for... G. parasuis When samples are mixed with PRRSV, they must be tested separately and cannot be screened simultaneously.
[0004] In recent years, recombinase polymerase amplification (RPA) isothermal technology has enabled rapid nucleic acid amplification at temperatures ranging from 37°C to 42°C. The CRISPR-Cas system further enhances this with its highly specific recognition and signal transduction capabilities, providing a new pathway for molecular diagnostics. However, current technologies lack primer combinations capable of simultaneously and specifically amplifying two pathogen targets in the same reaction system, resulting in insufficient specificity and low sensitivity when simultaneously recognizing two pathogen targets in isothermal amplification systems. Summary of the Invention
[0005] In order to develop a suitable G. parasuis This invention provides a primer combination, kit, method, and application for the dual detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus (PRRSV) to address the shortcomings of insufficient specificity and low sensitivity in the simultaneous recognition of two pathogen targets in existing isothermal amplification systems. The method achieves a sensitivity of 3.0 × 10⁻⁶. -8 The concentration is ng / μL, exhibiting good specificity. To achieve the above objectives, the present invention employs the following technical solution.
[0006] The present invention provides a primer combination for dual detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus, the primer combination comprising primer pair 1 and primer pair 2.
[0007] Primer pair 1 is a primer designed for the 16S rDNA gene of Haemophilus parasuis, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 1 ( G. parasuis -16S rDNA RPA primer-F or G. parasuis -F, 5'-AATTGCATTTCATACTGGGTTGCTAGAGTA-3') and downstream primers with nucleotide sequences as shown in SEQ ID NO.2 ( G. parasuis -16S rDNA RPA primer-R or G. parasuis -R, 5'-TATCTAATCCGTTTGCTCCCCACGCTTTC-3').
[0008] Primer pair 2 is targeted at porcine reproductive and respiratory syndrome virus. ORF7 Gene-designed primers, including upstream primers (PRRSV-) with nucleotide sequences such as SEQ ID NO.4. ORF7 RPA primer-F or PRRSV-F, 5'-TAATACGACTCACTATAGGGGCCAAATAACAACGGCAAGCAGCAGAAGAGA-3') and downstream primer (PRRSV-) with nucleotide sequence as shown in SEQ ID NO. 5. ORF7 RPA primer-R or PRRSV-R, 5'-GTGCAAGTCCCAGCGCCTTGATTAAAGGCGG-3').
[0009] The primer combination of the present invention targets the 16S rDNA gene of Haemophilus parasuis and PRRSV. ORF7 Specific primer pairs were designed for each gene, so that when the two pathogen targets were amplified synchronously under isothermal conditions, each primer pair only recognized the conserved region of its corresponding pathogen, avoiding cross-reaction. At the same time, the melting temperature, GC content and amplicon length of primer pair 1 and primer pair 2 were optimized and matched to ensure similar amplification efficiency in the same reaction system. This solved the defects of insufficient specificity and reduced sensitivity caused by primer competition or incompatible amplification conditions when the two pathogen targets were recognized synchronously in the isothermal amplification system, and achieved efficient and specific synchronous detection of two pathogens in mixed infection samples.
[0010] The present invention also provides a kit for dual detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus, including RT-RPA amplification reagent and CRISPR detection reagent.
[0011] The RT-RPA amplification reagent includes the primer combination, recombinase, single-stranded DNA binding protein, DNA polymerase, reverse transcriptase, and reaction buffer.
[0012] The CRISPR detection reagent includes Cas12a protein, Cas13a protein, crRNA targeting the 16S rDNA gene of Haemophilus parasuis, and a target for porcine reproductive and respiratory syndrome virus. ORF7 The gene's crRNA, fluorescent reporter molecule, T7 transcriptase, NTP, RNase inhibitor, and reaction buffer.
[0013] The nucleotide sequence of the crRNA targeting the 16S rDNA gene of Haemophilus parasuis is shown in SEQ ID NO.3 (5'-TAATTTCTACTAAGTGTAGATGCACATGAGCGTCAGTATTTTCC-3'); the nucleotide sequence of the crRNA targeting the porcine reproductive and respiratory syndrome virus is shown in SEQ ID NO.3 (5'-TAATTTCTACTAAGTGTAGATGCACATGAGCGTCAGTATTTTCC-3'). ORF7 The nucleotide sequence of the gene's crRNA is shown in SEQ ID NO.6 (5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGAUUAAAGGCGGUCUGGAUUGACGACAG-3').
[0014] Preferably, the fluorescent reporter molecule is a single-stranded DNA probe or a single-stranded RNA probe labeled with a fluorescent group and a quencher group.
[0015] Preferably, the nucleotide sequence of the single-stranded DNA probe labeled with a fluorescent group and a quencher group is as follows: 5'-TTTTTTT-3' (ssDNA), with FAM at the 5' end and BHQ1 at the 3' end.
[0016] The nucleotide sequence of the single-stranded RNA probe labeled with fluorescent and quencher groups is shown below.
[0017] 5'-UUUUUUU-3' (ssRNA), with ROX at the 5' end and BHQ2 at the 3' end.
[0018] Preferably, it also includes a positive control and a negative control.
[0019] The positive controls included Haemophilus parasuis 16S rDNA gene and porcine reproductive and respiratory syndrome virus. ORF7 Gene; the GenBank accession number for the Haemophilus parasuis 16S rDNA gene is AB004032.1, and the porcine reproductive and respiratory syndrome virus... ORF7The gene's GenBank accession number is NC 001961.1:14889-15260.
[0020] This invention provides a method for dual detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus, comprising the following steps: Sample processing: Extract total nucleic acid from the sample to be tested to obtain a nucleic acid mixture containing DNA and RNA.
[0021] Isothermal amplification: Using recombinase polymerase amplification technology, with the nucleic acid mixture as a template and the primer combination, RT-RPA amplification was performed under isothermal conditions to obtain amplification products of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus. Specifically, for Haemophilus parasuis (… G. parasuis Primer pair 1 was designed for the 16S rDNA gene, and RT-RPA amplification was performed under isothermal conditions to obtain Haemophilus parasuis ( G. parasuis The amplification products of the nucleic acid mixture were used; simultaneously, using the nucleic acid mixture as a template, the virus was targeted at porcine reproductive and respiratory syndrome virus (PRRSV). ORF7 Gene primer pair 2 was designed and RT-RPA amplification was performed under isothermal conditions to obtain the amplified product of porcine reproductive and respiratory syndrome virus (PRRSV).
[0022] CRISPR recognition and detection: The Haemophilus parasuis ( G. parasuis The amplification products of porcine reproductive and respiratory syndrome virus (PRRSV) are introduced into the Cas12a detection system, and the amplification products of PRRSV are introduced into the Cas13a detection system, and both are subjected to a CRISPR reaction in the same tube. The Cas12a detection system contains Cas12a protein, crRNA targeting the 16S rDNA gene of Haemophilus parasuis, and a fluorescent reporter molecule to recognize the DNA target and activate the trans-cleavage activity of Cas12a. The Cas13a detection system contains Cas13a protein, crRNA targeting the 16S rDNA gene of Haemophilus parasuis, and a fluorescent reporter molecule for recognizing the DNA target and activating the trans-cleavage activity of Cas12a. ORF7 The gene's crRNA, fluorescent reporter molecule, T7 transcriptase, and RNase inhibitor are used to transcribe the amplified PRRSV product DNA into RNA, which serves as a recognized RNA target and activates the trans-cleavage activity of Cas13a.
[0023] Signal detection: Detect the fluorescence signal generated by the above system, and determine the presence or absence of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus in the sample to be tested based on the presence or absence and brightness of the fluorescence color.
[0024] The method provided by this invention is to G. parasuisThe 16S rDNA gene is a DNA target, PRRSV ORF7 The genes are RNA targets, identified using Cas12a (DNA) and Cas13a (RNA) respectively. Combined with isothermal RT-RPA amplification, CRISPR, and fluorescence signal output, simultaneous qualitative and visual detection of two pathogens is achieved rapidly at an isothermal temperature. This invention optimizes the components and reaction conditions of the dual-enzyme system, achieving a sensitivity of 3.0 × 10⁻⁶. -8 It has a concentration of ng / μL, good specificity, and high detection efficiency; it also has a high concordance rate with clinical samples.
[0025] Preferably, the signal detection process also includes result interpretation, as follows: The reaction result shows green fluorescence, indicating that... G. parasuis Positive test result for PRRSV, negative test result for PRRSV; red fluorescence indicates a positive PRRSV test result. G. parasuis A negative result indicates a negative test; a yellow fluorescence (the range of yellow-green to orange-yellow is considered valid) indicates a negative result. G. parasuis Both PRRSV and PRRSV were positive; the reaction result was colorless, indicating a double negative result.
[0026] Preferably, the process for determining the presence or absence of Haemophilus parasuis and / or porcine reproductive and respiratory syndrome virus in the sample to be tested is as follows: If the fluorescence color is green, it indicates that Haemophilus parasuis is positive and porcine reproductive and respiratory syndrome virus is negative, thus determining that only Haemophilus parasuis exists in the sample to be tested.
[0027] If the fluorescence color is red, it indicates that the porcine reproductive and respiratory syndrome virus (PRRSV) is positive and Haemophilus parasuis is negative, thus determining that only PRRSV is present in the sample to be tested.
[0028] If the fluorescence color is in the range of yellow-green to orange-yellow, it indicates that Haemophilus parasuis and porcine reproductive and respiratory syndrome virus are both positive, thus determining that the sample to be tested contains both Haemophilus parasuis and porcine reproductive and respiratory syndrome virus.
[0029] If there is no fluorescence, it indicates that both Haemophilus parasuis and porcine reproductive and respiratory syndrome virus (PRRSV) are negative, thus determining that neither Haemophilus parasuis nor PRSV exists in the sample to be tested.
[0030] Preferably, the reaction conditions for the RT-RPA amplification are a temperature of 37℃~42℃ and a time of 10min~30min.
[0031] Preferably, the CRISPR reaction conditions are 35℃~39℃, 20min~60min.
[0032] Preferably, the Cas12a detection system and the Cas13a detection system further include T7 transcriptase, RNase inhibitor, and compatible buffer, respectively.
[0033] Preferably, the RPA amplification and CRISPR reaction are shown in Tables 1 and 2: Table 1 RPA reaction Table 2 CRISPR reaction Preferably, the fluorescent reporter molecule is a single-stranded DNA or single-stranded RNA probe labeled with a fluorescent group and a quencher group, wherein: The fluorescent reporter molecule in the Cas12a detection system is a single-stranded DNA probe, and its nucleotide sequence is shown below: 5'-TTTTTTT-3' (ssDNA), with FAM at the 5' end and BHQ1 at the 3' end.
[0034] The fluorescent reporter molecule in the Cas13a detection system is a single-stranded RNA probe, and its nucleotide sequence is shown below: 5'-UUUUUUU-3' (ssRNA), with ROX at the 5' end and BHQ2 at the 3' end.
[0035] The present invention also provides a detection primer and crRNA composition comprising primer pair 1, primer pair 2, crRNA targeting the 16S rDNA gene of Haemophilus parasuis, and crRNA targeting porcine reproductive and respiratory syndrome virus. ORF7 crRNA of the gene.
[0036] The primer pair 1 is a primer designed for the 16S rDNA gene of Haemophilus parasuis, which includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2.
[0037] Primer pair 2 is targeted at porcine reproductive and respiratory syndrome virus. ORF7 The genetically engineered primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO.4 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.5.
[0038] The nucleotide sequence of the crRNA targeting the 16S rDNA gene of Haemophilus parasuis is shown in SEQ ID NO. 3; the nucleotide sequence of the crRNA targeting porcine reproductive and respiratory syndrome virus is shown in SEQ ID NO. 3. ORF7 The nucleotide sequence of the gene's crRNA is shown in SEQ ID NO.6.
[0039] This invention also provides the primer combination or the kit for non-diagnostic purposes. G. parasuis Applications in PRRSV detection, pig farm monitoring, and epidemiological surveys, specifically at least one of the following applications: Pathogen screening for Haemophilus parasuis and porcine reproductive and respiratory syndrome virus in pig farms.
[0040] Detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus during port quarantine.
[0041] Epidemiological surveillance of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus.
[0042] Compared with the prior art, the present invention has the following beneficial effects: 1. In order to develop a suitable G. parasuis This invention provides a primer combination for the dual detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus (PRRSV), addressing the shortcomings of insufficient specificity and low sensitivity in existing isothermal amplification systems for the simultaneous recognition of two pathogen targets. This primer combination specifically recognizes the conserved region of the Haemophilus parasuis 16S rDNA gene and specifically recognizes PRRSV. ORF7 In conserved gene regions, the two primer pairs exhibit no cross-reactivity during synchronous amplification under isothermal conditions, and their amplification efficiencies are matched. This overcomes the shortcomings of existing technologies where unverified primer combinations lead to insufficient specificity and low sensitivity in isothermal dual detection. Furthermore, this invention optimizes the components and reaction conditions of the dual-enzyme system, achieving a sensitivity of 3.0 × 10⁻⁶. -8 With a concentration of ng / μL, good specificity, high concordance rate with clinical samples, and no need for a thermal cycler, it is compatible with on-site point-of-care testing (POCT) and suitable for pig farm screening, port quarantine and epidemiological monitoring. It enables efficient and specific simultaneous detection of two pathogens in mixed infection samples.
[0043] 2. This invention also provides a dual detection method for Haemophilus parasuis and porcine reproductive and respiratory syndrome virus based on this primer combination. The method provided by this invention includes the steps of RT-RPA amplification, T7 transcription, and CRISPR-Cas recognition and detection. RT-RPA amplification completes exponential amplification in a short time, increasing product yield and compensating for the shortcomings of insufficient sensitivity and low detection efficiency of CRISPR technology. It also contains reverse transcriptase, which can reverse transcribe the RNA template into DNA for amplification. T7 transcription completes the conversion of the product DNA to RNA, allowing the transcribed RNA to be recognized and cleaved by Cas13a. DNA sequences without the T7 promoter cannot be transcribed and are recognized and cleaved by Cas12a, completing the dual detection. The specific recognition of CRISPR-Cas solves the problem of non-specific amplification of RPA and enables signal conversion, making the detection results visible.
[0044] This invention effectively overcomes the inherent limitations of single technologies by synergistically integrating RPA and CRISPR-Cas systems. On the one hand, while RPA isothermal amplification is simple to operate, it is prone to non-specific amplification due to primer design or reaction conditions. This invention utilizes the sequence-specific recognition mechanism of Cas12a / Cas13a to perform secondary verification of the amplified products. Trans-cleavage activity is activated only when crRNA specifically binds to the target sequence, thereby significantly reducing the risk of false positives. On the other hand, the sensitivity of CRISPR detection alone is limited. This invention uses RPA pre-amplification to increase the initial template abundance, combined with the signal conversion effect of CRISPR, resulting in a significantly improved detection efficiency compared to CRISPR alone. At the same time, the Cas12a / Cas13a dual system enables parallel detection of Haemophilus parasuis and PRRSV in the same reaction tube, greatly improving detection throughput and ease of operation while ensuring specificity.
[0045] The method provided by this invention also has the following advantages: 1) Dual targets: Simultaneously detects DNA bacteria and RNA viruses, covering mixed infections.
[0046] 2) Constant temperature and rapid: No PCR instrument required, results in 60 minutes.
[0047] 3) High sensitivity: minimum 3.0×10 -8 ng / μL.
[0048] 4) High specificity: No cross-reactivity.
[0049] 5) Dual-channel: Fluorescence visualization, on-site friendly.
[0050] 6) Industrial applicability: This method can be industrially produced into lyophilized reagent kits, suitable for use in primary laboratories, mobile testing vehicles, and pig farms. G. parasuis Rapid screening, monitoring, and prevention of PRRSV have significant economic and social benefits.
[0051] 3. This invention discloses a Haemophilus parasuis strain based on RT-RPA-CRISPR-Cas12a / 13a ( G. parasuis This method, involving the dual detection of porcine reproductive and respiratory syndrome virus (PRRSV), belongs to the field of molecular diagnostic technology for animal diseases. G. parasuis The 16S rDNA gene is a DNA target, PRRSV ORF7The genes are RNA targets, identified using Cas12a (DNA) and Cas13a (RNA) respectively. Combined with isothermal RT-RPA amplification, CRISPR, and fluorescence signal output, simultaneous qualitative and visual detection of two pathogens is achieved rapidly at an isothermal temperature. This invention optimizes the components and reaction conditions of the dual-enzyme system, achieving a sensitivity of 3.0 × 10⁻⁶. -8 The method exhibits high specificity (ng / μL) and high concordance rate with clinical samples. It requires no thermal cycling instrument, is compatible with point-of-care testing (POCT), and is suitable for pig farm screening, port quarantine, and epidemiological monitoring. Attached Figure Description
[0052] Figure 1 In this invention G. parasuis Optimization results of dual RPA with PRRSV.
[0053] Figure 2 In this invention G. parasuis Results of temperature optimization for dual RPA reaction with PRRSV.
[0054] Figure 3 In this invention G. parasuis Optimization of the best primer ratio for the PRRSV dual RPA system.
[0055] Figure 4 In this invention G. parasuis Optimization results of T7 RNA Polymerase in the PRRSV dual RPA-CRISPR-Cas system; among which: A represents the ultraviolet results image plus the chemiluminescence imaging system results image; B is the result image from the real-time PCR instrument.
[0056] Figure 5 In this invention G. parasuis Optimization results of the RNase inhibitor in the PRRSV dual RPA-CRISPR-Cas system; among which: A represents the ultraviolet results image plus the chemiluminescence imaging system results image; B is the result image from the real-time PCR instrument.
[0057] Figure 6 In this invention G. parasuis Optimization results of RPA products in the PRRSV dual RPA-CRISPR-Cas system; among which: A represents the ultraviolet results image plus the chemiluminescence imaging system results image; B is the result image from the real-time PCR instrument.
[0058] Figure 7 In this invention G. parasuisOptimization of Cas12a and Cas13a content in the PRRSV dual RPA-CRISPR-Cas system; wherein: A is the UV result graph; B is the result image from the real-time PCR instrument.
[0059] Figure 8 In this invention G. parasuis Optimization of reaction conditions with PRRSV dual RPA-CRISPR-Cas.
[0060] Figure 9 for G. parasuis Both PRRSV and PRRSV underwent conventional PCR amplification at 3.0 × 10⁻⁶. 0 ng / μL ~ 3.0 × 10 -7 For templates with a concentration of ng / μL, the limit of detection was 3.0 × 10⁻⁶. -5 ng / μL; where: A is G. parasuis Standard PCR sensitivity testing; B represents the sensitivity detection of PRRSV using standard PCR. M: DNA Standard Marker 2000; 1:3.0×10 0 ng / μL; 2: 3.0×10 -1 ng / μL; 3: 3.0×10 -2 ng / μL; 4: 3.0×10 -3 ng / μL; 5: 3.0×10 -4 ng / μL; 6: 3.0×10 -5 ng / μL; 7: 3.0×10 -6 ng / μL; 8: 3.0×10 -7 ng / μL; 9: negative control.
[0061] Figure 10 In this invention G. parasuis Sensitivity test of dual RPA-CRISPR-Cas detection with PRRSV; where: A represents the ultraviolet results image plus the chemiluminescence imaging system results image; B is the result image from the real-time PCR instrument; 1:3.0×10 -3 ng / μL; 2: 3.0×10 -4 ng / μL; 3: 3.0×10 -5 ng / μL; 4: 3.0×10 -6 ng / μL; 5: 3.0×10 -7 ng / μL; 6: 3.0×10 -8ng / μL; 7: 3.0×10 -9 ng / μL; 8: negative control.
[0062] Figure 11 In this invention G. parasuis The specificity detection of the PRRSV dual RT-RPA-CRISPR-Cas system showed only target positivity, with no visible color in the control; among which: A and C are the UV results; B and D are the results of the real-time PCR instrument.
[0063] Figure 12 The clinical sample fluorescence results in this invention clearly distinguish between single positive, double positive, and negative results; a total of 168 samples were tested, and the images show that the dual RPA-CRISPR-Cas detection method was used. G. parasuis Fifteen samples were positive (samples 3, 14, 21, 27, 37, 41, 53, 64, 73, 80, 91, 96, 100, 105, and 109), and four samples were positive for PRRSV (samples 19, 37, 78, and 96). Two of these samples were double-positive (samples 37 and 96), and the remaining colorless tubes contained double-negative samples. Detailed Implementation
[0064] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.
[0065] Example 1: Reagent Preparation (1) Standard plasmid: G. parasuis The template (Haemophilus parasuis 16S rDNA gene, GenBank accession number AB004032.1) was extracted using the Seville Bacterial Genomic DNA Extraction Kit, strain ( G. parasuis This was provided by the Liaoning Provincial Key Laboratory of Zoonotic Diseases Research, College of Animal Science and Medicine, Shenyang Agricultural University. See “Preliminary Study on Bivalent Subunit Vaccine of Haemophilus parasuis and Actinobacillus pleuropneumoniae LpxC-linker-ApxIVA”, Shenyang Agricultural University, Wang Linxi.
[0066] The PRRSV-N plasmid was developed by Shanghai Sangon Biotech Co., Ltd. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) ORF7 Sequence (Porcine Reproductive and Respiratory Syndrome Virus) ORF7 The gene (with GenBank accession number NC 001961. 1:14889-15260) was obtained by ligating it into the pUC57 vector.
[0067] The above-mentioned synthesized plasmids were extracted using the Tiangen Plasmid Mini-Prep Kit. The RNA template for PRRSV was provided by the Liaoning Provincial Key Laboratory of Zoonotic Diseases, College of Animal Science and Medicine, Shenyang Agricultural University, as described in "Establishment and Preliminary Application of CRISPR-Cas13a Detection Method for Seneca Virus", Shenyang Agricultural University, Wu Yuhan.
[0068] Among them, the Actinobacillus pleuropneumoniae, Streptococcus suis, Escherichia coli, Salmonella, Staphylococcus aureus, Proteus mirabilis, and porcine epidemic diarrhea virus used in the specificity test of the RT-RPA-CRISPR-Cas detection method were all preserved by the Liaoning Provincial Key Laboratory of Zoonotic Diseases Research, College of Animal Science and Medicine, Shenyang Agricultural University. Among them, *Actinobacillus pleuropneumoniae* is referred to in "Preliminary Study on Bivalent Subunit Vaccine of Haemophilus parasuis and Actinobacillus pleuropneumoniae LpxC-linker-ApxIVA", Shenyang Agricultural University, Wang Linxi; *Staphylococcus aureus* is referred to in "Preparation of ASMA-TAT and Its Antibacterial Effect on Staphylococcus aureus and Simulated Rumen Transmission Study", Shenyang Agricultural University, Hong Yu; *Streptococcus suis* is referred to in "Preparation of Monoclonal Antibody and Identification of Antigenic Epitope of Streptococcus suis Type 2 SSU05-0474 Protein", Shenyang Agricultural University, Yang Baoling; *Escherichia coli* and *Salmonella* are referred to in "Screening and Identification Analysis of Brucella Surface Antigen Nucleic Acid Aptamers", Shenyang Agricultural University, Wang Hao; *Proteus mirabilis* is referred to in "Isolation, Identification and Drug Sensitivity Analysis of Proteus mirabilis in Cooked Meat Products", *Advances in Animal Medicine*, Yang Junjie et al.; and porcine epidemic diarrhea virus (PEDV) is referred to in "Establishment and Application of Double Droplet Digital PCR Detection Method for PEDV and TGEV", Shenyang Agricultural University, Wu Nan.
[0069] The porcine circovirus type 2 vaccine strain and the pseudorabies virus vaccine strain were purchased from Shenyang Agricultural University Weimin Animal Husbandry Technology Service Co., Ltd.
[0070] (2) Transcription of RNA: PRRSV-N plasmid was transcribed into RNA using the Beijing Lamborghini T7 High Yield RNA Synthesis Kit, following the instructions. Then, Trizol purification was performed using the Nanjing Novizan RNAisolater Total RNA Extraction Reagent, following the instructions. ORF7 RNA fragments (Porcine reproductive and respiratory syndrome virus) ORF7 (crRNA of the gene).
[0071] (3) crRNA: in vitro transcription / synthesis and purification (steps as above).
[0072] Among them, the nucleotide sequence of crRNA targeting the 16S rDNA gene of Haemophilus parasuis ( G. parasuis -crRNA-Cas12a) as shown in SEQ ID NO.3: 5'-TAATTTCTACTAAGTGTAGATGCACATGAGCGTCAGTATTTTCC-3'.
[0073] Targeting porcine reproductive and respiratory syndrome virus ORF7 The nucleotide sequence of the gene's crRNA (PRRSV-crRNA-Cas13a) is shown in SEQ ID NO. 6: 5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACGAUUAAAGGCGGUCUGGAUUGACGACAG-3'.
[0074] (4) Probe: synthesized by Shanghai Sangon Biotech Co., Ltd. After receiving it, centrifuge at 4000 rpm for 1 min, add enzyme-free water according to the tube markings to dissolve the dry powder and prepare a storage solution for later use.
[0075] The fluorescent reporter molecule in the Cas12a detection system is a single-stranded DNA probe, and its nucleotide sequence (ssDNA-FAM-BHQ1) is shown below: 5'-TTTTTTT-3' (ssDNA), with FAM at the 5' end and BHQ1 at the 3' end.
[0076] The fluorescent reporter molecule in the Cas13a detection system is a single-stranded RNA probe, and its nucleotide sequence (ssRNA-ROX-BHQ2) is shown below: 5'-UUUUUUU-3' (ssRNA), with ROX at the 5' end and BHQ2 at the 3' end.
[0077] Example 2: Dual RPA Optimization 1. Double RPA amplification time: 10 min, 15 min, 20 min, 25 min, and 30 min, respectively, at 39℃. The reaction system is as shown in Table 1. After the reaction, add 8 μL of 6× Loading buffer and continue heat denaturation at 56℃ for 5 min. Then, take 5 μL of the product and perform electrophoresis on a 2% agarose gel for 40 min at 100V. Determine the optimal amplification time based on the brightness of the bands in the results.
[0078] Table 1 RPA reaction See results Figure 1When there is no significant difference in band brightness, a shorter reaction time of 10 minutes is selected.
[0079] 2. Temperatures for double RPA amplification: 35℃, 37℃, 39℃, 41℃, 43℃. The reaction system is as shown in Table 1. After the reaction, add 8μL of 6×Loading buffer to the product and continue heat denaturation at 56℃ for 5min. Then, take 5μL of the product and perform electrophoresis on a 2% agarose gel for 40min at 100V. Determine the optimal amplification temperature based on the brightness of the bands in the results.
[0080] See results Figure 2 Since there was no significant difference in band brightness, the subsequent reaction was carried out at 37°C, the same temperature as the CRISPR reaction.
[0081] 3. Primer ratio: G. parasuis The primer ratios with PRRSV were 1:1, 1:3, and 3:1, and the reaction system was as shown in Table 1. After the reaction, the product was added to 8 μL of 6× Loading buffer and heat-denatured at 56 °C for 5 min. 5 μL of the product was then aspirated into a 2% agarose gel for electrophoresis at 100 V for 40 min. The optimal primer ratio was determined based on the brightness of the bands.
[0082] See results Figure 3 : G. parasuis The optimal primer ratio for PRRSV is 3:1.
[0083] Primer pair 1 includes G. parasuis -16S rDNA RPA primer-F and G. parasuis -16S rDNARPA primer-R.
[0084] G. parasuis -16S rDNA RPA primer-F ( G. parasuis The nucleotide sequence of -F) is as shown in SEQ ID NO.1: 5'-AATTGCATTTCATACTGGGTTGCTAGAGTA-3'.
[0085] G. parasuis -16S rDNA RPA primer-R ( G. parasuis The nucleotide sequence of (-R) is shown in SEQ ID NO.2: 5'-TATCTAATCCGTTTGCTCCCCACGCTTTC-3'.
[0086] Primer pair 2 includes PRRSV- ORF7 RPA primers-F and PRRSV- ORF7 RPA primer-R.
[0087] PRRSV- ORF7 The nucleotide sequence of RPA primer-F (PRRSV-F) is shown in SEQ ID NO.4: 5'-TAATACGACTCACTATAGGGGCCAAATAACAACGGCAAGCAGCAGAAGAGA-3'.
[0088] PRRSV- ORF7 The nucleotide sequence of RPA primer-R (PRRSV-R) is shown in SEQ ID NO.5: 5'-GTGCAAGTCCCAGCGCCTTGATTAAAGGCGG-3'.
[0089] Example 3: Optimization of the two-enzyme system 1. T7 RNA Polymerase and NTPs: 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL and 1.0 μL, the reaction system is shown in Table 2. Adjust the amount of T7 RNA Polymerase and NTPs in Table 2, and record the results using a UV and chemiluminescence imaging system and a real-time PCR instrument to determine the optimal content.
[0090] Table 2 CRISPR reaction See results Figure 4 The optimal concentration of T7 RNA Polymerase and NTPs is 0.2 μL.
[0091] 2. RNase Inhibitor: 0.2 μL, 0.4 μL, 0.6 μL, 0.8 μL and 1.0 μL. The reaction system is shown in Table 2. Adjust the amount of RNase Inhibitor in Table 2, and record the results using a UV and chemiluminescence imaging system and a real-time PCR instrument to determine the optimal content.
[0092] See results Figure 5 The optimal concentration of RNase Inhibitor is 0.4 μL.
[0093] 3. Add 1 μL, 2 μL, 3 μL, 4 μL and 5 μL of the dual RPA product to the CRISPR system, respectively. The reaction system is shown in Table 2. Adjust the amount of RPA product added, and record the results using a UV and chemiluminescence imaging system and a real-time PCR instrument to determine the optimal content.
[0094] See results Figure 6 The minimum amount of RPA product added is 3 μL.
[0095] 4. Cas12a and Cas13a: Cas12a (0.5 μL) / Cas13a (0.5 μL), Cas12a (0.5 μL) / Cas13a (1.0 μL), Cas12a (1.0 μL) / Cas13a (0.5 μL), Cas12a (1.0 μL) / Cas13a (1.0 μL). The reaction system is shown in Table 2. The content of the two Cas proteins was adjusted, and the results were recorded by UV and real-time PCR instruments to determine the optimal amount of the two enzymes.
[0096] See results Figure 7 The optimal content of the two is Cas12a (1.0 μL) / Cas13a (0.5 μL).
[0097] Example 4: Optimization of RPA-CRISPR reaction conditions use G. parasuis The bacterial suspensions (TSB tryptone soybean broth medium) and PRRSV-N plasmid glycerol bacteria (LB liquid medium) were subjected to double RPA for 10 min.
[0098] Dual CRISPR-Cas reaction temperatures: 39℃, 37℃, 35℃, palm temperature.
[0099] Dual CRISPR-Cas reaction times: 20 min, 30 min, 40 min, 50 min, 60 min.
[0100] Controls were set up: RPA reaction for 30 min and CRISPR-Cas reaction for 60 min at 39℃, 37℃, 35℃, and palm temperature. The fluorescence colors of the dual RPA-CRISPR-Cas detection method under different reaction times and temperatures were compared under UV light to determine the optimal reaction time and temperature.
[0101] See results Figure 8 : G. parasuis Yellow fluorescence can be observed in the bacterial culture after RPA for 10 min + CRISPR for 50 min at 37℃, and it can also be achieved with palm temperature.
[0102] in, G. parasuis The method for obtaining the bacterial culture is as follows: G. parasuis The product can be obtained by isothermal shaking culture at 37°C for 12 hours in tryptone soybean broth medium. G. parasuis Bacterial solution.
[0103] Example 5: Sensitivity Test 10x gradient dilution G. parasuis With PRRSV template (3.0×10) -3 ng / μL – 3.0 × 10 -9The minimum detection limit of this method was determined by using these templates (ng / μL) for dual detection with RPA-CRISPR-Cas, and the presence of visible fluorescence color under ultraviolet light was used to determine the detection limit of the method.
[0104] See results Figure 10 : G. parasuis Both PRRSV and PRRSV reached 3.0 × 10 -8 ng / μL. At 3.0 × 10⁻⁶ 0 ng / μL – 3.0 × 10 -7 Two templates, each in ng / μL, were used in a standard PCR assay, and the results were visualized using agarose gel electrophoresis.
[0105] See results Figure 9 : G. parasuis Both PRRSV and PRRSV reached 3.0 × 10 -5 Compared to PCR, the method established in this study is 1000 times more sensitive (ng / μL).
[0106] The primer sequences used in PCR detection are shown in Table 3. The amplification system for PCR detection is shown in Table 4. The amplification procedure for PCR detection is shown in Table 5.
[0107] Table 3 Primer Sequences Table 4 Amplification System Table 5 Amplification Procedure Example 6: Specificity Test Detection G. parasuis For PRRSV double-positive templates, Streptococcus suis, porcine circovirus type 2, Actinobacillus pleuropneumoniae, and pseudorabies virus, only the double-positive template shows fluorescence (orange-yellow) under ultraviolet light, while the others are completely negative. There are also single-positive templates... G. parasuis The method was demonstrated to be able to distinguish between single and mixed pathogens by detecting single PRRSV positive templates and double positive templates. Figure 11 ).
[0108] The above results demonstrate that this method has good specificity.
[0109] Example 7: Clinical Sample Testing 168 clinical samples were collected and compared with the results of PCR testing. (See attached image) Figure 12 : (1) This method: G. parasuis Of the 15 positive samples, 4 were PRRSV positive, including 2 double-positive samples.
[0110] (2) Compliance rate: Positive compliance rate G. parasuisThe success rate was 93.75%, and the PRRSV success rate was 100%; the negative compliance rate was... G. parasuis The percentage was 99.35%, and the PRRSV was 100%.
[0111] Example 8: Fluorescence Detection (1) Fluorescence: FAM channel = G. parasuis =Green, ROX channel=PRRSV=Red.
[0112] As can be seen from the above, the established G. parasuis A dual RPA-CRISPR-Cas detection method with PRRSV was developed, with reaction conditions of 10 min RPA and 50 min CRISPR. It exhibits good specificity and sensitivity, with a limit of detection of 3.0 × 10⁻⁶. - 8 ng / μL. In clinical sample testing, the results of dual RPA-CRISPR-Cas and PCR methods showed high consistency and a high positive concordance rate. G. parasuis The success rate was 93.75%, and the PRRSV success rate was 100%; the negative compliance rate was... G. parasuis The percentage was 99.35%, and the PRRSV was 100%.
[0113] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.
[0114] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.
Claims
1. A primer combination for dual detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus, characterized in that, The primer pair includes primer pair 1 and primer pair 2; Primer pair 1 is a primer designed for the 16S rDNA gene of Haemophilus parasuis, comprising an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 1 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 2; Primer pair 2 is targeted at porcine reproductive and respiratory syndrome virus. ORF7 The gene-designed primers include an upstream primer with a nucleotide sequence as shown in SEQ ID NO.4 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.
5.
2. A kit for dual detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus, characterized in that, Includes RT-RPA amplification reagents and CRISPR detection reagents; The RT-RPA amplification reagent includes the primer combination, recombinase, single-stranded DNA binding protein, DNA polymerase, reverse transcriptase, and reaction buffer described in claim 1. The CRISPR detection reagent includes Cas12a protein, Cas13a protein, crRNA targeting the 16S rDNA gene of Haemophilus parasuis, and a target for porcine reproductive and respiratory syndrome virus. ORF7 The gene's crRNA, fluorescent reporter molecule, T7 transcriptase, NTP, RNase inhibitor, and reaction buffer; The nucleotide sequence of the crRNA targeting the 16S rDNA gene of Haemophilus parasuis is shown in SEQ ID NO.3; the nucleotide sequence of the crRNA targeting porcine reproductive and respiratory syndrome virus is shown in SEQ ID NO.
3. ORF7 The nucleotide sequence of the crRNA of the gene is shown in SEQ ID NO.
6.
3. The reagent kit according to claim 2, characterized in that, The fluorescent reporter molecule is a single-stranded DNA probe or a single-stranded RNA probe labeled with a fluorescent group and a quencher group.
4. The reagent kit according to claim 3, characterized in that, The nucleotide sequence of the single-stranded DNA probe labeled with fluorescent and quencher groups is shown below: 5'-TTTTTTT-3', with its 5' end marked FAM and its 3' end marked BHQ1; The nucleotide sequence of the single-stranded RNA probe labeled with fluorescent and quencher groups is shown below: 5'-UUUUUUU-3', with ROX marked at the 5' end and BHQ2 marked at the 3' end.
5. The reagent kit according to claim 4, characterized in that, It also includes positive and negative controls; The positive controls included Haemophilus parasuis 16S rDNA gene and porcine reproductive and respiratory syndrome virus. ORF7 The gene; the GenBank accession number for the Haemophilus parasuis 16S rDNA gene is AB004032.1, and the porcine reproductive and respiratory syndrome virus... ORF7 The gene's GenBank accession number is NC 001961.1:14889-15260.
6. A method for dual detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus, characterized in that, Includes the following steps: Total nucleic acids are extracted from the sample to be tested to obtain a mixture of nucleic acids containing DNA and RNA; Using the nucleic acid mixture as a template, and employing the primer combination described in claim 1, RT-RPA amplification was performed under isothermal conditions to obtain amplification products of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus. The amplification product of the Haemophilus parasuis was introduced into the Cas12a detection system, and the amplification product of the porcine reproductive and respiratory syndrome virus was introduced into the Cas13a detection system. The two were then subjected to a CRISPR reaction in the same tube. The presence or absence of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus in the sample to be tested is determined based on the fluorescence color and brightness. The Cas12a detection system comprises the Cas12a protein, crRNA targeting the 16S rDNA gene of Haemophilus parasuis, and a fluorescent reporter molecule; the Cas13a detection system comprises the Cas13a protein and a target for porcine reproductive and respiratory syndrome virus. ORF7 The gene's crRNA, fluorescent reporter molecules, T7 transcriptase, and RNase inhibitor.
7. The method according to claim 6, characterized in that, The process for determining the presence or absence of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus in the sample to be tested is as follows: If the fluorescence color is green, it indicates that Haemophilus parasuis is positive and porcine reproductive and respiratory syndrome virus is negative, thus determining that only Haemophilus parasuis exists in the sample to be tested; If the fluorescence color is red, it indicates that the porcine reproductive and respiratory syndrome virus is positive and the Haemophilus parasuis is negative, thus determining that only porcine reproductive and respiratory syndrome virus exists in the sample to be tested; If the fluorescence color is in the range of yellow-green to orange-yellow, it indicates that Haemophilus parasuis and porcine reproductive and respiratory syndrome virus are both positive, and thus it is determined that Haemophilus parasuis and porcine reproductive and respiratory syndrome virus are present in the sample to be tested. If there is no fluorescence, it indicates that both Haemophilus parasuis and porcine reproductive and respiratory syndrome virus (PRRSV) are negative, thus determining that the sample to be tested does not contain Haemophilus parasuis and PRSV.
8. The method according to claim 6, characterized in that, The reaction conditions for the RT-RPA amplification were a temperature of 37℃~42℃ and a time of 10min~30min.
9. The method according to claim 6, characterized in that, The CRISPR reaction conditions are 35℃~39℃, 20min~60min.
10. The application of the primer combination according to claim 1 or the kit according to any one of claims 2 to 5, characterized in that, For at least one of the following applications: Pathogen screening for Haemophilus parasuis and porcine reproductive and respiratory syndrome virus in pig farms; Detection of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus during port quarantine; Epidemiological surveillance of Haemophilus parasuis and porcine reproductive and respiratory syndrome virus.