Multiplex fluorescent quantitative PCR detection kit and application

Abstract

The present application relates to the technical field of virus detection in biotechnology, in particular to a multiplex fluorescent quantitative PCR detection kit and application. The reaction solution in the kit contains a pair of universal primers and four specific TaqMan probes, and is marked with different fluorescent groups, so that one reaction system can simultaneously identify PRRSV classical strain, highly pathogenic strain, NADC30-like strain and NJ-1106R vaccine strain, and has the characteristics of strong specificity and high sensitivity. The multiplex fluorescent PCR improves the detection efficiency and reduces the detection cost, and provides technical support for simultaneous differential diagnosis of PRRSV strain types in clinic.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology in biotechnology, and particularly to a multiplex quantitative PCR detection kit and its application. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS) is a reproductive disorder and respiratory infectious disease in pigs caused by the porcine reproductive and respiratory syndrome virus (PRRSV). It mainly causes abortion, stillbirth, and mummified fetuses in pregnant sows, and piglets exhibit respiratory disorders.

[0003] Based on the sequence differences in the PRRSV genome, it is divided into the European genotype, represented by Lelystad virus (LV), and the American genotype, represented by the classic strain VR-2332. The two strains are similar in morphology and physicochemical properties, but differ in antigenicity and cell tropism. Significant differences exist in the gene sequences of the European and American strains, particularly in the Nsp2 gene, where the corresponding amino acid homology is only about 32%.

[0004] Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) exhibits significant variations compared to previously circulating PRRSV strains. A key marker of this variant is the deletion of 30 amino acids in the Nsp2 gene (positions 482 and 538–566, based on the classic strain VR-2332). The NADC 30-like strain is characterized by a deletion of 131 amino acids in the Nsp2 region (111 amino acids at positions 323–433, 1 amino acid at position 482, and 19 amino acids at positions 534–552, with a deletion pattern of "111+1+19", based on the classic strain VR-2332).

[0005] In 2011, piglets at a large-scale pig farm in Nanjing exhibited mild respiratory problems. Lungs and lymph nodes from piglets showing mild high fever and cough were collected, and a moderately virulent PRRSV strain, NJ-1106 (GenBank accession number JX880029), was isolated. Identification using reverse transcription-polymerase chain reaction (RT-PCR), indirect immunofluorescence assay (IFA), and whole-genome sequencing confirmed the isolate as PRRSV. After 150 generations of passage, an artificially attenuated strain, NJ-1106R, was obtained as a vaccine candidate strain. Compared to classic PRRSV strains (based on VR-2332), this strain, in addition to the deletion of 30 common amino acids (positions 482 and 538–566) in the Nsp2 gene, also has a deletion of 144 amino acids at positions 621–764.

[0006] Currently, there is no multiplex quantitative PCR detection method that can simultaneously detect PRRSV classic strains, highly pathogenic strains, NADC30-like strains, and NJ-1106R vaccine strains. Summary of the Invention

[0007] This invention provides a multiplex quantitative PCR detection method capable of simultaneously identifying classic strains, highly pathogenic strains, NADC30-like strains, and NJ-1106R vaccine strains of porcine reproductive and respiratory syndrome virus.

[0008] This invention is achieved through the following operations:

[0009] A multiplex quantitative PCR detection kit, wherein the reaction solution of the multiplex quantitative PCR detection kit contains the following primers and probes:

[0010] PRRSV-F: 5'-TTATTCTCCGGGTGATTGGG-3',

[0011] PRRSV-R: 5'-CTACACTCTGGCGAGTCAAACT-3',

[0012] P1: JOE-TGCCTCTTTTTATGTTACAGTTACCCAGCCTTTG-BHQ1,

[0013] P2: FAM-TGCCTCTTTCTATGTTACAGTTACCCATTCTTCG-BHQ1,

[0014] P3: Cy5-TGCCTCTTTCTGTGTTACAGTTATCCTGCGTTTG-BHQ3,

[0015] P4:ROX-TGCCTCTTTCTGTGTTACAGCTACCCATTCTTTG-BHQ2.

[0016] The multiplex quantitative PCR detection kit is preferably further comprising 2×TaqMan mix in the reaction solution.

[0017] The multiplex quantitative PCR detection kit preferably contains the following reaction solution: 10 μL of 2×TaqMan mix, 1 μL of 10 μM PRRSV-F, 1 μL of 10 μM PRRSV-R, 0.8 μL of 5 μM P1, 1 μL of 5 μM P2, 1 μL of 5 μM P3, 0.8 μL of 5 μM P4, 0.5 μL of the cDNA to be tested, and ddH2O to make up to 20 μL.

[0018] The application of the described multiplex quantitative PCR detection kit in the detection of porcine reproductive and respiratory syndrome virus.

[0019] In the aforementioned application, the reaction solution is preferably placed in a corresponding real-time PCR instrument, and the PCR reaction conditions are: 95℃, 3 min; 95℃, 10 s, 63℃, 35 s, 40 cycles.

[0020] In the application described above, the porcine reproductive and respiratory syndrome virus is preferably a classic strain, a highly pathogenic strain, a NADC30-like strain, or an NJ-1106R vaccine strain.

[0021] The preferred application selection criteria are as follows:

[0022] If a quantitative fluorescence amplification curve of P1 appears and the Ct value is ≤35, then the sample to be tested contains a classic strain.

[0023] If a quantitative fluorescence amplification curve of P2 appears and the Ct value is ≤35, then the sample to be tested contains a highly pathogenic strain.

[0024] If a quantitative fluorescence amplification curve of P3 appears and the Ct value is ≤35, then the sample to be tested contains a NADC30-like strain.

[0025] If a quantitative fluorescence amplification curve of P4 appears and the Ct value is ≤35, then the sample to be tested contains the NJ-1106R vaccine strain.

[0026] The preferred application selection criteria also include:

[0027] If 35 < Ct ≤ 40 for the tested sample, repeat the experiment after increasing the sample loading volume. If the Ct value ≤ 40 is satisfied, make a judgment according to the above content. If the Ct value > 40 or no signal is shown, the sample does not contain classical strains, highly pathogenic strains, NADC30-like strains or NJ-1106R vaccine strains of PRRSV.

[0028] Principle and technical solution of the present invention: Based on the Nsp2 gene sequence differences among classical strains, highly pathogenic strains, NADC30-like strains and NJ-1106R vaccine strains of PRRSV, a pair of universal primers are designed in the gene conserved region, and the differential sites of the amplified fragments of various strains are further analyzed. For the differential sites, 4 specific TaqMan probes targeting classical strains, highly pathogenic strains, NADC30-like strains and NJ-1106R vaccine strains of PRRSV are designed, and are labeled with JOE, FAM, Cy5 and ROX respectively. The multiplex fluorescence quantitative PCR reaction system is optimized to achieve the effect of simultaneously identifying multiple viruses in one reaction system.

[0029] The 4 probes are designed for the differential sites of Nsp2 of different strains respectively, where P1 targets classical PRRSV strains, P2 targets highly pathogenic PRRSV strains, P3 targets NADC30-like PRRSV strains, and P4 targets PRRSV NJ-1106R vaccine strains.

[0030] The detection method is as follows:

[0031] For PRRSV strains and other RNA virus samples, use the TRIZOL method or a commercially available RNA extraction kit to extract RNA, and reverse transcribe with a commercially available reverse transcription kit to obtain cDNA.

[0032] Configure the PCR reaction system in the corresponding reaction tubes of a four-channel or more four-channel fluorescence quantitative PCR instrument according to the following table:

[0033]

[0034] PRRSV multiplex fluorescence quantitative PCR conditions:

[0035] Place the PCR reaction tubes in the corresponding fluorescence quantitative PCR instrument, and the PCR reaction conditions are: 95 °C, 3 min; 95 °C, 10 s, 63 °C 35 s (collect fluorescence signals of different channels), 40 cycles.

[0036] Advantages of the present invention:

[0037] (1) This invention analyzes the differences in Nsp2 gene sequences of PRRSV classical strains, highly pathogenic strains, NADC30-like strains and NJ-1106R vaccine strains. A pair of universal primers is designed for the conserved Nsp2 gene sequences of different strains, which can simultaneously amplify different PRRSV strains. Furthermore, four specific TaqMan probes are designed for the differential sites in this amplification region and labeled with different fluorescent groups, so that one reaction system can simultaneously identify PRRSV classical strains, highly pathogenic strains, NADC30-like strains and NJ-1106R vaccine strains;

[0038] (2) The TaqMan probe-based quantitative PCR method has the characteristics of high specificity and high sensitivity. Multiplex fluorescent PCR improves detection efficiency and reduces detection costs, providing technical support for the simultaneous identification and diagnosis of clinical PRRSV strains. Attached Figure Description

[0039] Figure 1 To ensure the specificity of the probe P1 corresponding to the classic PRRSV strain in this invention, the JOE channel...

[0040] Figure 2 To ensure the specificity of the probe P2 corresponding to the highly pathogenic PRRSV strain of this invention, the FAM channel...

[0041] Figure 3 To enhance the specificity of the probe P3 corresponding to the NADC30 PRRSV strain of this invention, the Cy5 channel...

[0042] Figure 4 To enhance the specificity of the PRRSV NJ-1106R vaccine strain corresponding to probe P4, the ROX channel...

[0043] Figure 5 To determine the sensitivity of the real-time PCR method corresponding to probe P1 in this invention, the JOE channel and the concentration of cDNA template from the classic PRRSV strain were 1×10⁻⁶. 1 -1×10 8 Copy / μL,

[0044] Figure 6 To determine the sensitivity of the quantitative real-time PCR method corresponding to probe P2 in this invention, the concentration of cDNA template from the FAM channel and the highly pathogenic PRRSV strain was 1×10⁻⁶. 1 -1×10 8 Copy / μL,

[0045] Figure 7 To determine the sensitivity of the quantitative real-time PCR method corresponding to probe P3 in this invention, the concentration of cDNA template from the NADC30PRRSV strain in the Cy5 channel was 1×10⁻⁶. 1-1×10 8 Copy / μL,

[0046] Figure 8 To determine the sensitivity of the real-time PCR method corresponding to probe P4 in this invention, the concentrations of the ROX channel and the cDNA template of the PRRSV NJ-1106R vaccine strain were 1×10⁻⁶. 1 -1×10 8 Copy / μL. Detailed Implementation

[0047] The technical solution of this application will be described in detail below with reference to specific embodiments.

[0048] Example 1: Materials and Methods for PRRSV Multiplex Quantitative PCR

[0049] 1.1 Design and synthesis of primers and probes

[0050] By comparing and analyzing the differences in the Nsp2 gene sequence of classic PRRSV strains, highly pathogenic strains, NADC30-like strains, and NJ-1106R vaccine strains, a pair of universal primers was designed in the conserved gene region to simultaneously amplify the Nsp2 gene fragments of different PRRSV strains. Further analysis of the differential sites in the amplified fragments of each strain was conducted. Based on these differential sites, four specific TaqMan probes were designed for the classic PRRSV strains, highly pathogenic strains, NADC30-like strains, and NJ-1106R vaccine strain, respectively labeled with JOE, FAM, Cy5, and ROX, for the detection of PRRSV by multiplex quantitative PCR. The primer and probe sequences are shown in Table 1.

[0051]

[0052] 1.2 Sample Preparation

[0053] Samples of PRRSV classic strain, highly pathogenic strain, NADC30-like strain, and NJ-1106R vaccine strain stored at -80℃ were removed, brought to room temperature, and then used for later use.

[0054] Samples of swine fever virus (CSFV) and swine influenza virus (SIV) stored at -80℃ were removed, brought to room temperature, and used as virus controls.

[0055] If the clinical sample is a pathological tissue, take about 2 g of the pathological tissue, cut it into small pieces, add 300 μL of sterile PBS and grind it, then freeze and thaw it 3 times, centrifuge at 10,000 rpm for 5 min, and take 200 μL of the supernatant and put it into a sterile EP tube for later use; if the clinical sample is a cotton swab, place the cotton swab in a 5 mL sterile EP tube with 300 μL of sterile PBS, freeze and thaw it 3 times, repeatedly blow and agitate the cotton swab, and take 200 μL of the supernatant and put it into a sterile EP tube for later use.

[0056] 1.3 Viral RNA Extraction and Reverse Transcription

[0057] Take 200 μL of supernatant from PRRSV classic strain, highly pathogenic strain, NADC30-like strain, NJ-1106R vaccine strain, CSFV sample, SIV sample, cotton swab or pathogen sample, extract RNA using TRIZOL method or commercial RNA extraction kit, and reverse transcribe using commercial reverse transcription kit to obtain cDNA.

[0058] 1.4 Optimization of PRRSV multiplex real-time PCR reaction conditions

[0059] Take 0.5 μL of cDNA from steps 1.2 and 1.3, and prepare the PCR reaction system according to the table below in the reaction tubes of a four-channel or higher real-time PCR instrument:

[0060]

[0061] The reaction conditions, including primer and probe concentrations and annealing temperature, were optimized to screen out the best conditions for quantitative real-time PCR. The optimal reaction system is shown in Table 2. The optimal reaction conditions were: 95 ℃ for 3 min; 95 ℃ for 10 s, 63 ℃ for 35 s (collecting fluorescence signals from different channels), for 40 cycles.

[0062] Analysis of the results of this invention:

[0063] (1) System effectiveness analysis: The reaction wells corresponding to the negative control did not show typical quantitative fluorescence amplification curves of JOE, FAM, Cy5 and ROX; the reaction wells corresponding to the positive control showed typical quantitative fluorescence amplification curves of JOE, FAM, Cy5 and ROX, and the Ct value was <35.

[0064] (2)Result determination: If there is a typical amplification curve of JOE fluorescence quantification in the sample reaction well and the Ct value ≤ 35, it is determined that the sample contains the classical PRRSV strain; if there is a typical amplification curve of FAM fluorescence quantification in the sample reaction well and the Ct value ≤ 35, it is determined that the sample contains the highly pathogenic PRRSV strain; if there is a typical amplification curve of Cy5 fluorescence quantification in the sample reaction well and the Ct value ≤ 35, it is determined that the sample contains the NADC30-like PRRSV strain; if there is a typical amplification curve of ROX fluorescence quantification in the sample reaction well and the Ct value ≤ 35, it is determined that the sample contains the PRRSV NJ-1106R vaccine strain.

[0065] If 35 < Ct ≤ 40 for the tested sample, the sample loading amount needs to be increased and the test repeated. If, after repetition, the Ct value ≤ 40 and there is a typical amplification curve of the corresponding fluorescence, the type of PRRSV strain is determined according to the above principles; if, after repetition, the Ct value > 40 or no signal is shown, it is determined that there is no corresponding fluorescence signal and the sample does not contain the corresponding PRRSV strain.

[0066] Example 2: Specificity test

[0067] According to the amplification system of the present invention, cDNA samples of the classical PRRSV strain, highly pathogenic strain, NADC30-like strain, NJ-1106R vaccine strain, CSFV, and SIV were used as templates to verify the specificity of the system.

[0068] For the classical PRRSV strain of the present invention, the corresponding probe P1 only detected the cDNA signal of the classical PRRSV strain in the JOE channel, while the cDNA samples of the highly pathogenic PRRSV strain, NADC30-like strain, NJ-1106R vaccine strain, CSFV, and SIV were all negative ( Figure 1 ); similarly, for the highly pathogenic PRRSV strain, the corresponding probe P2 only detected the cDNA signal of the highly pathogenic PRRSV strain in the FAM channel ( Figure 2 ); for the NADC30-like PRRSV strain, the corresponding probe P3 only detected the cDNA signal of the NADC30-like PRRSV strain in the Cy5 channel ( Figure 3 ); for the PRRSV NJ-1106R vaccine strain, the corresponding probe P4 only detected the cDNA signal of the PRRSV NJ-1106R vaccine strain in the ROX channel ( Figure 4 ). This indicates that the method has strong specificity and no cross-reactivity with other PRRSV strains or other porcine disease pathogens.

[0069] Example 3: Sensitivity test

[0070] cDNA samples from the classic PRRSV strain, highly pathogenic strain, NADC30-like strain, and NJ-1106R vaccine strain were taken and diluted to 1×10⁻⁶ with sterile double-distilled water. 1 -1×10 8 According to the amplification system of this invention, 0.5 μL of diluted cDNA was added to each tube for multiplex quantitative PCR detection, with 3 replicates, to determine the sensitivity of the multiplex quantitative PCR reaction.

[0071] The lower limit of detection of the cDNA signal of the classic PRRSV strain by probe P1 in the JOE channel is 10. 2 copy( Figure 5 The probe P2 corresponding to the highly pathogenic PRRSV strain was used to detect the cDNA signal of the highly pathogenic PRRSV strain in the FAM channel. Figure 6 The probe P3 corresponding to the NADC30 PRRSV strain was used to detect the cDNA signal of the NADC30 PRRSV strain in the Cy5 channel. Figure 7 The PRRSV NJ-1106R vaccine strain cDNA signal was detected by probe P4 in the ROX channel. Figure 8 The lower limit of all of them can reach 10. 2 The copy number indicates that this method has high sensitivity and can be used for clinical detection of PRRSV strains.

[0072] Example 4: Blind Sample Detection

[0073] Five samples each of pathogenic material and swabs from the classic PRRSV strain, the highly pathogenic strain, the NADC30-like strain, and the NJ-1106R vaccine strain, along with five samples of pathogenic material and swabs from samples without PRRSV, were taken as blind samples. RNA was extracted and reverse transcribed from each sample. Blind sample detection was performed according to the amplification system of this invention. The detection results were compared with the original information from the pathogenic material or swabs to calculate the accuracy of the blind sample detection.

[0074] Blind sample testing was conducted using the multiplex quantitative PCR method of this invention. Five samples each of PRRSV classical strain pathological material and swabs showed typical quantitative PCR amplification curves in the JOE channel, with Ct values ​​all less than 35, achieving a 100% accuracy rate. Five samples each of highly pathogenic strain pathological material and swabs showed typical quantitative PCR amplification curves in the FAM channel, with Ct values ​​all less than 35, achieving a 100% accuracy rate. Five samples each of NADC30-like strain pathological material and swabs showed typical quantitative PCR amplification curves in the Cy5 channel, with Ct values ​​all less than 35, achieving a 100% accuracy rate. Five swabs of NJ-1106R vaccine strain showed typical quantitative PCR amplification curves in the ROX channel, with Ct values ​​all less than 35, achieving a 100% accuracy rate. Five samples each of pathological material and swabs without PRRSV showed no typical quantitative PCR amplification curves in any channel, achieving a 100% accuracy rate. The overall results showed a 100% agreement with the original records, indicating that this method is suitable for identifying PRRSV strain types in clinical samples.

[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments. Any changes, modifications, combinations, substitutions, or simplifications made without departing from the spirit and principle of the present invention should be considered as equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. Use of a multiplex fluorescence quantitative PCR detection kit for differentiating classical strains, highly pathogenic strains, NADC30-like strains or NJ-1106R vaccine strains of porcine reproductive and respiratory syndrome virus for non-diagnostic purposes, The reaction solution in the multiplex fluorescence quantitative PCR detection kit contains the following primers and probes: PRRSV-F: 5’-TTATTCTCCGGGTGATTGGG-3’, PRRSV-R: 5’-CTACACTCTGGCGAGTCAAACT-3’, P1: JOE-TGCCTCTTTTTATGTTACAGTTACCCAGCCTTTG-BHQ1, P2: FAM-TGCCTCTTTCTATGTTACAGTTACCCATTCTTCG-BHQ1, P3: Cy5-TGCCTCTTTCTGTGTTACAGTTATCCTGCGTTTG-BHQ3, P4: ROX-TGCCTCTTTCTGTGTTACAGCTACCCATTCTTTG-BHQ2; The reaction solution is placed in a corresponding fluorescence quantitative PCR instrument, and the PCR reaction conditions are: 95°C, 3 min; 95°C, 10 s, 63°C, 35 s, 40 cycles.

2. The application according to claim 1, characterized in that... The reaction solution in the multiplex fluorescence quantitative PCR detection kit also contains 2×TaqMan mix.

3. The application according to claim 1, characterized in that... The reaction solution in the multiplex fluorescence quantitative PCR detection kit contains 10 μL of 2×TaqMan mix, 1 μL of 10 μM PRRSV-F, 1 μL of 10 μM PRRSV-R, 0.8 μL of 5 μM P1, 1 μL of 5 μM P2, 1 μL of 5 μM P3, and 0.8 μL of 5 μM P4.

4. The application according to claim 1, wherein If a fluorescence quantitative amplification curve of P1 appears and the Ct value ≤ 35, the test sample contains a classical strain; If a fluorescence quantitative amplification curve of P2 appears and the Ct value ≤ 35, the test sample contains a highly pathogenic strain; If a fluorescence quantitative amplification curve of P3 appears and the Ct value ≤ 35, the test sample contains an NADC30-like strain; If a fluorescence quantitative amplification curve of P4 appears and the Ct value ≤ 35, the test sample contains an NJ-1106R vaccine strain.

5. The application according to claim 4, wherein If 35 < Ct ≤ 40 for the test sample, repeat the test after increasing the sample loading amount. If the Ct value ≤ 40 is satisfied, make a determination according to the steps of claim 4. If the Ct value > 40 or no curve is shown, the sample does not contain classical strains, highly pathogenic strains, NADC30-like strains or NJ-1106R vaccine strains.

6. The application according to claim 4, characterized in that... The multiplex quantitative PCR detection kit has a detection sensitivity of 10 for classic strains, highly pathogenic strains, NADC30-like strains, and NJ-1106R vaccine strains. 2 Copy / μL.

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