Human enterovirus A-D group broad-spectrum detection reagent based on fluorescent quantitative PCR (Polymerase Chain Reaction) technology

Abstract

The invention discloses a human enterovirus A-D group broad-spectrum detection reagent based on a fluorescent quantitative PCR (Polymerase Chain Reaction) technology. The human enterovirus A-D group broad-spectrum detection reagent comprises a specific upstream primer group, a specific downstream primer group and a specific probe group, by adopting a fluorescent quantitative detection method, not only can the viruses be quickly screened, but also the load of the viruses can be quantified, and the fluorescent quantitative detection method has great advantages for researching the infection state of the viruses and the influence of the viruses on the disease progress. The reagent has the characteristics of high specificity, high sensitivity, high repeatability, high reproducibility, simplicity in operation, low time consumption and the like, and can greatly improve the detection efficiency of the enterovirus and reduce the detection cost when being applied to large-scale clinical screening, and the detection result can provide important reference for formulating an individualized treatment scheme, so that the reagent has a wide application prospect. Therefore, the application has important value and significance for clinical rapid screening and diagnosis and improvement of treatment effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of enterovirus detection, and in particular relates to a broad-spectrum detection reagent for human enterovirus AD group based on fluorescence quantitative PCR technology. Background Art

[0002] Enteroviruses (EVs) belong to the genus Enterovirus in the family Picornaviridae. Their genome consists of a single-stranded, positive-sense RNA (SSTR) approximately 7.4 kb in length, encoding 11 viral proteins (VP1-VP4, 2A-2C, 3A-3D). Currently, EVs are classified into 12 enteroviruses (Group AL) and three rhinoviruses (RV-A, RV-B, and RV-C). Of all enteroviruses, only rhinoviruses and Group AD enteroviruses can infect humans. Group AC enteroviruses include poliovirus (PV; Group C), echovirus (E; Group B), coxsackievirus A (CVA; Groups A and C), and coxsackievirus B (CVB; Group B), while Group D enteroviruses include EV-D68, EV-D70, EV-D94, EV-D111, and EV-D120. Enteroviruses primarily enter the human body through the digestive or respiratory tract, proliferating in the respiratory epithelium and pharyngeal or intestinal lymph nodes, and are transmitted through the fecal-oral route and the respiratory tract. Human enterovirus infection can cause respiratory, digestive, neurological, and reproductive system diseases, including acute flaccid myelitis, hand, foot, and mouth disease, acute hemorrhagic conjunctivitis, infertility, neonatal sepsis, encephalitis, myocarditis, acute otitis media, herpangina, and diarrhea. It has become a public health concern and has attracted widespread clinical attention. Data show that enterovirus infection can affect people of all ages, primarily children, and large-scale, periodic epidemics are highly likely to occur. It is important to note that enteroviruses can easily cause severe infections in immunocompromised individuals or newborns, and some enteroviruses can easily cause nosocomial outbreaks of neonatal infections. Therefore, detection of enterovirus infection is crucial in clinical diagnosis and treatment.

[0003] Poliovirus, the first enterovirus discovered, primarily causes poliomyelitis, commonly known as "polio," which can lead to paralysis in children in severe cases. There are three serotypes of poliovirus: PV1-3, with PV1 being the most pathogenic. The virus primarily attacks the nervous system, particularly the motor neurons in the anterior horns of the spinal cord, causing muscle weakness, paralysis, and even death.

[0004] Echoviruses, with 30 confirmed serotypes, primarily infect children under five years of age. They can cause multisystem diseases, including nonspecific rashes such as maculopapular rash, acute hemorrhagic conjunctivitis, myopericarditis, type 1 diabetes, sepsis, and gastroenteritis. Existing research suggests that echoviruses also have potential gonadotropin-tropic properties, potentially affecting human reproductive function through mechanisms such as the blood-testis barrier, the blood-egg barrier, and interference with the immune microenvironment.

[0005] Coxsackieviruses are divided into group A (23 serotypes) and group B (6 serotypes), with significant differences in their clinical pathogenicity. Group A viruses primarily cause hand, foot, and mouth disease (types A16, A6, and A10) and herpangina (types A2, A4-A6, A8, and A10), primarily affecting children aged 3 to 10 years. Group B viruses, on the other hand, are more likely to cause myocarditis (types B3 and B5) and severe neonatal infections (types B1-B5). Studies have also shown that group B coxsackieviruses can cause orchitis and decreased ovarian function, leading to infertility. Notably, coxsackievirus A21 (CVA21) has recently been linked to acute flaccid paralysis, with a neurotoxic mechanism similar to that of poliovirus.

[0006] Currently, the main detection methods for human enteroviruses include enzyme-linked immunosorbent assay (ELISA), loop-mediated isothermal amplification (LAMP), virus isolation and culture, and real-time fluorescence quantitative PCR. Although ELISA has been used for clinical detection and screening of enterovirus antigens or antibodies, the results of antigen or antibody tests cannot accurately determine the viral infection status, resulting in significant limitations in practical application. Furthermore, LAMP technology, due to its accuracy and complexity, has significantly limited its widespread application in clinical diagnosis. Furthermore, virus isolation and culture are time-consuming and its sensitivity is affected by sample quality, resulting in significant limitations in practical application. In contrast, real-time fluorescence quantitative PCR, which uses fluorescent signals to monitor the PCR amplification process in real time, can accurately quantify viral load and has become an important method for laboratory testing and clinical diagnosis of pathogen infections. However, due to the significant similarity of genomic nucleotides among different types of human enteroviruses, the specific detection of a single enterovirus group using traditional single primers and probes is difficult. Consequently, existing detection reagents can only detect a very small number of enterovirus genotypes. Currently, there is no clinically available method based on fluorescence quantitative PCR that can detect a broad spectrum of enterovirus AD groups. To address this issue, the present invention specifically designs primer and probe sets in appropriate ratios based on target gene frequency to achieve broad-spectrum detection of enteroviruses belonging to the AD group, which can infect humans. (Although rhinoviruses can also infect humans, this method excludes rhinoviruses due to their lower overall prevalence.) While this method cannot specifically distinguish specific genotypes of enteroviruses belonging to the AD group, it covers all genotypes of the human enterovirus AD group and their associated serotypes, ensuring the accuracy of test results. Furthermore, combined with viral gene amplification, sequencing, and evolutionary analysis, it can ultimately distinguish and identify specific viral types. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention provides a broad-spectrum detection reagent for human enterovirus AD group based on fluorescent quantitative PCR technology, which includes a specific upstream primer set, a specific downstream primer set and a specific probe set; and also includes other conventional commercially available reagents for fluorescent quantitative PCR detection; The specific upstream primer set consists of the following primers: EVsFM1: 5'-TGDYRGTGRCTGCGYTGGCG-3'; EVsFM2: 5'-GTGTAGATCAGGYCGATGAGTCA-3'; EVsFM3: 5'-GTGTAGCTTRGGYYGATGAGTCTG-3'; EVsFM4: 5'-GCCYGCGTGGCTGCCT-3'; EVsFM5: 5'-ACTAGTYTGGTCGATGAGGCT-3'; EVsFM6: 5'- GACAWGGTGBGAAGARYCTAYTG-3'; EVsFM7: 5'-TGDYGYAGCCYGCGTGGTGC-3'; EVsFM8: 5'- CCRRYKGTAGCTCTGRRDRAT-3'; EVsFM9: 5'-CTGTYTTGGCGTTTCGCTC-3'; The primer set was prepared by mixing the above primers (concentration before mixing was 10 μM) in the following ratios: EVsFM1 5 μL, EVsFM2 4 μL, EVsFM3 3 μL, EVsFM4 2 μL, EVsFM5 1 μL, EVsFM6 2 μL, EVsFM7 1 μL, EVsFM8 1 μL, EVsFM9 1 μL; The specific downstream primer set consists of the following primers: EVsRM1: 5'-CTGTYTTGGCGTTTCGCTC-3'; EVsRM2: 5'- GAAACACGGDYWYCCAAAGTAGT-3'; The above primers (concentration before mixing was 10 μM) were mixed in the following ratios to prepare the primer set: EVsRM1 8 μL, EVsRM2 2 μL; The specific probe group consists of the following probes: EVsPM1: 5'-HEX- TCCGGCCCCTGAATGYGGCTAAYYYYAA-BHQ1-3'; EVsPM2: 5'-HEX- TCCGGYYCCTGAATGYGGCTAAYCYWA-BHQ1-3'; EVsPM3: 5'-HEX- TCCGGCCCCTGAATGYGGHYAATCCYAAC-BHQ1-3'; EVsPM4: 5'-HEX- CCGGRGCCGTGAATGCTGCTAATCCYAAC-BHQ1-3'; The above probes (concentration before mixing was 5 μM) were mixed in the following ratio to prepare the probe set: EVsPM1 3 μL, EVsPM2 3 μL, EVsPM3 3 μL, and EVsPM4 1 μL.

[0008] The method for using the above-mentioned fluorescent quantitative PCR detection reagent is as follows: 1. Collect clinical stool samples from patients, collect and record patient information during sampling, register them, store them at low temperature during transportation, and store them at -80℃; 2. Use the Tiangen Virus Genomic DNA / RNA Extraction Kit to extract the viral RNA genome from the stool sample; 3. Using the RNA extracted in step 2 as a template, employ the human enterovirus-specific primer set and probe set described above, and perform fluorescence quantitative PCR according to the optimized reaction procedure and reaction system of the Acryl One-Step Kit (Ag11713). The results are determined based on the Ct value. The reaction procedure is as follows: reverse transcription at 42°C for 5 min, pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, and annealing and extension at 58°C for 30 s, for 40 cycles. Fluorescence signals are collected during the extension phase of each cycle.

[0009] 4. Interpretation of test results includes the following: A. Determination of the validity of the amplification curve: (1) Amplification curve shape: It should be a standard "S" curve, which should be smooth without jagged fluctuations and have a normal plateau phase; (2) Threshold line setting requirements: located in the middle of the exponential growth period, not too high or too low; (3) Quality control requirements: The Ct value of the negative control / no-template control should be UNDEF, and the difference in Ct values ​​between replicate wells should be <0.5 (otherwise, retesting or re-extraction of nucleic acid is required).

[0010] B. Result interpretation criteria

[0011] Special Notes It is recommended that samples in the suspicious range be retested for confirmation; UNDEF indicates that the sample concentration is below the detection limit and is uniformly judged as negative; all positive results must meet the validity criteria of the amplification curve.

[0012] Compared with previous detection methods, the present invention has the following advantages: 1. Specificity validation of the method of the present invention revealed no cross-reaction between the enteroviruses and other pathogens, and good specificity. Sensitivity validation of the method revealed that the minimum detection limit for enteroviruses could reach 100 copies / μL, indicating that the method has high sensitivity. Repeatability validation of the method revealed that the coefficient of variation (CV) for both intra-batch and inter-batch repeatability was less than 3%, indicating good repeatability and reproducibility of the method. 2. The method of the present invention has the advantages of high sensitivity, strong specificity, good repeatability and reproducibility, and provides an effective technical means for the clinical diagnosis of human enterovirus AD group. This method is characterized by high efficiency and accuracy, and is suitable for clinical infection diagnosis, epidemiological monitoring and dynamic analysis of viral load, which can significantly improve detection efficiency. Through large-scale application in clinical patients, the promotion of this technology will help to clarify the molecular mechanisms of diseases caused by enterovirus infection and provide an important basis for clinical diagnosis and treatment.

[0013] In summary, by utilizing the principles of qPCR technology and adopting a primer set and probe set strategy, a broad-spectrum detection of AD group enteroviruses and their related serotypes that can infect humans can be achieved. At the same time, the test results can provide etiological evidence for the formulation of clinical treatment plans, and help implement isolation, treatment, and prevention measures. It has important value and significance for the rapid diagnosis and treatment of clinical enterovirus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the amplification curve result of human enterovirus with different probe concentrations; Figure 2 This is the amplification curve result of human enterovirus at different annealing temperatures; Figure 3 This is the specific experimental result of fluorescence quantitative PCR; Figure 4 Verify the amplification curve results for the linear interval of human enterovirus by fluorescent quantitative PCR; Figure 5 This is the standard curve result of enterovirus fluorescence quantitative PCR. DETAILED DESCRIPTION

[0015] To further illustrate the technical means and effects of the present invention, the following specific embodiments are used to further illustrate the technical solutions of the present invention, but the present invention is not limited to the scope of the embodiments. It should be noted that the materials used in the following examples are not limited to the above-mentioned materials and can be replaced by other similar materials. If the specific conditions of the instruments are not specified, they should be used under conventional conditions or according to the conditions recommended by the manufacturer. Those skilled in the art should have relevant knowledge of using conventional materials and instruments. Unless otherwise specified, the methods in the examples are all conventional methods. Example 1: Design of specific primers and probes 1. Download the full-length genome nucleic acid sequence of human enterovirus from the NCBI website; 2. Use Mega11 software to perform nucleic acid sequence alignment to identify the conserved gene region of the human enterovirus genome, and use Primer Select software to design specific primer sets and probe sets for the human enterovirus 5'UTR gene; 3. Primer and probe BLAST evaluation: The nucleotide sequences of the primers and probes designed were compared again using the BLAST search function on the NCBI website to select primers and probe sequences with high specificity. The nucleotide sequences of the specific primer set and probe set for the human enterovirus AD group are shown in Table 1.

[0016] Table 1 Sequences of primer sets and probe sets specific for human enterovirus AD group .

[0017] Example 2: Establishment of enterovirus fluorescence quantitative PCR method 1. Plasmid construction and verification The enterovirus-specific gene sequence (SEQ ID NO: 1) was ligated with the pUC57 vector to synthesize the enterovirus-positive plasmid GD1P1. Plasmid construction was completed by Sangon Biotech Co., Ltd. The synthesized plasmid was activated in glycerol bacteria, and the plasmid was extracted and sequenced for verification. The concentration was measured using an ultraviolet spectrophotometer, and the copy number of the plasmid was calculated based on the length and concentration of each plasmid. The calculation formula is as follows:

[0018] The copy number results are shown in Table 2 below: Table 2 Copy numbers of human enterovirus-positive plasmids ; 2. Optimization of fluorescence quantitative reaction system The above plasmid standards were diluted tenfold (10 10 -10 1 According to the initial reaction system and reaction conditions recommended in the instructions of the one-step kit (Ag11713) of Acryl, 10 9 ~10 2 Real-time fluorescence quantitative PCR was performed with a plasmid standard of 1000 copies / μL. Based on the experimental results, a positive standard with a Ct value between 15 and 30 cycles, high fluorescence intensity, and a good amplification curve was selected as a template for reaction system optimization. Finally, a concentration of 10 7 The positive standard with 100 copies / μL was used as a template for system optimization.

[0019] The final concentrations of the upstream or downstream primers of each gene in the reaction system were set to 100nM, 200nM, 300nM, and 400nM, respectively. The "matrix method" was used to optimize the primer concentrations, and the Ct and Std values ​​were shown in Table 3 below. The final concentrations of the probes in the reaction system were set to 50nM, 100nM, 150nM, and 200nM, respectively. The probe concentrations were optimized, and the Ct and Std values ​​were shown in Table 4 below. The amplification curve results were shown in Figure 1 As shown; the annealing temperature was set to 50℃, 52℃, 54℃, 56℃, 58℃, and 60℃ respectively for annealing temperature optimization. The amplification curve results are shown in Figure 2 The average Ct and Std values ​​were calculated by triplicate for each gradient. Based on the following principles: a relatively small Ct value, a well-defined S-shaped amplification curve, high and concentrated fluorescence intensity, and minimal primer dimers, the optimal concentrations of upstream and downstream primers in the reaction system were determined to be 300 nM; the optimal probe concentration was 150 nM; and the optimal annealing temperature was 58°C.

[0020] Table 3 Ct and Std values ​​of human enterovirus at different primer concentrations

[0021] Table 4 Ct and Std values ​​of different probe concentrations for human enterovirus

[0022] The final fluorescence quantitative PCR reaction system was determined as follows: 2×One Step RT-qPCR Buffer Ⅱ (probe) 10μL, pro Taq HS DNA Polymerase (5U / μL) 0.4μL, Evo M-MLV RTase Enzyme Mix II 0.4 μL, EVs-F / R 0.6 μL each, EVs-P 0.6 μL, template 1 μL, and add water to make up to 20 μL.

[0023] The fluorescence quantitative PCR reaction program was finally determined as follows: reverse transcription at 42°C for 5 min, pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 58°C for 30 s, 40 cycles; fluorescence was collected during the extension stage of each cycle.

[0024] Example 3: Evaluation of the specificity, linear range, sensitivity, and repeatability of the primer-probe fluorescence quantitative PCR of the present invention 1. Specificity verification of fluorescent quantitative PCR The viral genome extracts of two DNA viruses (herpes simplex virus-HSV, adenovirus-ADV) and five RNA viruses (rhinovirus-HRV, human immunodeficiency virus-HIV, dengue virus-DENV, rotavirus-RV, astrovirus-AstV) with clinically verified nucleic acid positive were used as well as 10 7 A plasmid standard with 100 copies / μL was used as a template, and sterile water was added as a negative control. The specificity of fluorescent quantitative PCR was verified according to the reaction system and procedure optimized in Example 2.

[0025] The results are as follows Figure 3 As shown, except for the enterovirus positive plasmid standard, the other viruses were not amplified. There was no cross-reaction between the enterovirus detection primers and other viral nucleic acid templates, indicating that the method has good specificity.

[0026] 2. Verification of the linear interval of fluorescent quantitative PCR The positive plasmid standard was diluted to 10 by 10-fold serial dilution method. 9 -10 2 copies / μL, sterile water was used as a negative control, and a specific primer set and probe set for enterovirus were used to verify the linear interval of the present invention according to the optimized reaction system and procedure; The results are as follows Figure 4 As shown, the amplification curves are 10 from left to right. 9 copies / μL, 10 8 copies / μL, 10 7 copies / μL, 10 6 copies / μL, 10 5 copies / μL, 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL; a standard curve was established with the logarithm of template concentration as the horizontal axis and the number of cycles as the vertical axis, such as Figure 5 As shown in the figure, the standard curve constructed based on the highly conserved sequence of enterovirus has an amplification efficiency of 87.26%, R 2 It is 0.9983, indicating a good linear relationship; 3. Sensitivity verification of fluorescence quantitative PCR After linear interval verification, the minimum detection limit of enterovirus was preliminarily determined to be 10 2 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1With three gradients of 100 copies / μL as templates and sterile water as negative control, 20 repeated experiments were performed using the fluorescence quantitative PCR detection method according to the optimized reaction system and reaction procedure. If the detection rate could stably reach more than 95%, it would be finally determined as the minimum detection limit.

[0027] The results are shown in Table 5 below. The minimum detection limit of the detection system of the present invention for human enterovirus is 100 copies / μL.

[0028] Table 5 Results of the lowest detection limit for human enterovirus

[0029] 4. Fluorescence quantitative PCR repeatability verification In order to verify the repeatability of the fluorescence quantitative PCR detection method, a concentration gradient of 10 8 -10 2 A plasmid standard containing 100 copies / μL of plasmid was used as a positive template. Detection was performed using a Biori FQD-96a real-time fluorescence quantitative PCR instrument. The reaction system and amplification procedure were the same as in Example 2. Fluorescence signals were collected during the extension phase of each cycle to verify intra- and inter-batch reproducibility. Within-batch repetitions were performed: seven gradients, each with three replicates, repeated once; between-batch repetitions were performed: seven gradients, each with three replicates, repeated three times. The Ct value was recorded for each step, and the coefficient of variation (CV) was calculated using the following formula to evaluate reproducibility. A coefficient of variation (CV) of less than 3% for both intra- and inter-batch results, calculated from the Ct values, indicates good reproducibility and repeatability.

[0030]

[0031] The results of the repeatability verification are shown in Table 6 below. The results show that the coefficient of variation CV is less than 3%, indicating that the repeatability and reproducibility of the present invention are good; Table 6 Repeatability verification results of human enterovirus .

[0032] Example 4: Fluorescence quantitative PCR detection of clinical samples 10g stool samples were collected from 20 patients clinically diagnosed with positive human enterovirus nucleic acid and placed in sterile EP tubes. The patient information was carefully verified, the samples were labeled, and the samples were registered. The samples were stored at 4°C during transportation and at -80°C until nucleic acid extraction. 1. Nucleic acid extraction RNA was extracted using the Tiangen Virus Genomic DNA / RNA Extraction Kit. The specific steps are as follows: a. Before using the Tiangen Virus Genomic DNA / RNA Extraction Kit, add 70 mL and 17 mL of anhydrous ethanol to the rinse buffer PW and buffer GD, respectively; b. Sample aliquoting: Weigh 0.2 g of fecal sample into a sterile EP tube, add 1 ml of normal saline, vortex and centrifuge at 12,000 rpm for 5 min, aliquot 200 μL of clarified fecal supernatant into a new EP tube, and add 20 μL of Protein K; c. Add 200 μL of the prepared Carrier RNA working solution to the EP tube, vortex and incubate in a 56°C water bath for 15 minutes, then briefly centrifuge and add 250 μL of anhydrous ethanol. Vortex for 15 seconds and let stand for 5 minutes. A flocculent precipitate will form. d. Transfer the flocculent precipitate and solution mixture to an adsorption column, centrifuge at 8000 rpm for 1 min, discard the waste liquid, add 500 μL of buffer GD, centrifuge at 8000 rpm for 1 min, and discard the waste liquid; place the adsorption column in a collection tube; e. Add 600 μL of rinse solution PW, let it stand for 2 minutes, then centrifuge at 8000 rpm for 1 minute and discard the waste liquid. Repeat this step twice; f. Add 500 μL of anhydrous ethanol, centrifuge at 8000 rpm for 1 min, discard the waste liquid, and then centrifuge at 12000 rpm for 3 min, discard the waste liquid; h. Place the adsorption column in a new enzyme-free centrifuge tube, add 45 μL of RNase-Free ddH2O to the middle of the adsorption membrane, let it stand at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 3 minutes to collect the nucleic acid solution at the bottom of the tube. Aliquot according to the experimental situation and store at -20°C for later use.

[0033] 2. Fluorescence quantitative PCR detection The nucleic acid extracted in step 2 was used as a template. The reaction system was similar to that in Example 2. The amplification procedure was as follows: reverse transcription at 42°C for 5 min, pre-denaturation at 95°C for 30 s, denaturation at 95°C for 5 s, annealing and extension at 58°C for 30 s, 40 cycles. Fluorescence was collected during the extension phase of each cycle. The detection results are shown in Table 7 below: Table 7 Fluorescence quantitative PCR detection results

[0034] As can be seen from the above table, the reagent of the present invention has the advantages of high sensitivity, strong specificity, good repeatability and simple operation. It can quickly and accurately detect human enterovirus, thereby improving the clinical screening speed of enterovirus.

Claims

1. A broad-spectrum detection reagent for human enterovirus AD group based on fluorescent quantitative PCR technology, characterized in that: It includes a specific upstream primer set, a specific downstream primer set and a specific probe set; The specific upstream primer set consists of the following primers: EVsFM1: 5'-TGDYRGTGRCTGCGYTGGCG-3'; EVsFM2: 5'-GTGTAGATCAGGYCGATGAGTCA-3'; EVsFM3: 5'-GTGTAGCTTRGGYYGATGAGTCTG-3'; EVsFM4: 5'-GCCYGCGTGGCTGCCT-3'; EVsFM5: 5'-ACTAGTYTGGTCGATGAGGCT-3'; EVsFM6: 5'- GACAWGGTGBGAAGARYCTAYTG-3'; EVsFM7: 5'-TGDYGYAGCCYGCGTGGTGC-3'; EVsFM8: 5'- CCRRYKGTAGCTCTGRRDRAT-3'; EVsFM9: 5'-CTGTYTTGGCGTTTCGCTC-3'; The specific downstream primer set consists of the following primers: EVsRM1: 5'-CTGTYTTGGCGTTTCGCTC-3'; EVsRM2: 5'- GAAACACGGDYWYCCAAAGTAGT-3'; The specific probe group consists of the following probes: EVsPM1: 5'-HEX- TCCGGCCCCTGAATGYGGCTAAYYYYAA-BHQ1-3'; EVsPM2: 5'-HEX- TCCGGYYCCTGAATGYGGCTAAYCYWA-BHQ1-3'; EVsPM3: 5'-HEX- TCCGGCCCCTGAATGYGGHYAATCCYAAC-BHQ1-3'; EVsPM4: 5'-HEX- CCGGRGCCGTGAATGCTGCTAATCCYAAC-BHQ1-3'.

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