Primer combination, kit and application for detecting bovine norovirus based on rtrpa-crispr cas12a

Abstract

This invention discloses primer combinations, kits, and applications for detecting bovine norovirus based on RTRPA-CRISPR / Cas12a, belonging to the field of biotechnology. 1. This invention combines reverse transcription recombinase polymerase amplification (RT-RPA) technology with the CRISPR / Cas12a system, successfully applying it to the detection of bovine norovirus (BNoV). This method integrates the high efficiency of isothermal amplification with the ultra-high specificity of the CRISPR / Cas system, constructing a novel BNoV nucleic acid detection technology system. This provides a practical new solution for rapid on-site diagnosis of BNoV. Furthermore, the optimized core components, crRNA and primers, work synergistically to efficiently and specifically amplify the BNoV target region, resulting in excellent detection accuracy. In addition, the detection method established in this invention does not rely on expensive thermal cycling equipment; it only requires a constant temperature incubator or water bath and a portable fluorescence detector. The entire detection process is shorter than the traditional qPCR method, and has a wider range of applications.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to primer combinations, kits, and applications for detecting bovine norovirus based on RTRPA-CRISPRCas12a. Background Technology

[0002] Bovine norovirus (BNoV) is a major pathogen causing diarrhea in calves. Infection can lead to intestinal damage and severe watery diarrhea, resulting in stunted growth and development, and causing significant economic losses to the cattle industry. This virus belongs to the genus Norovirus of the family Caliciviridae. It is a non-enveloped, single-stranded, positive-sense RNA virus, with known genotypes including GIII.1 and GIII.2. Since its first report in my country, BNoV has been detected in cattle herds in multiple provinces, showing a widespread prevalence.

[0003] Currently, there is a lack of efficient, rapid, and field-applicable technologies for the early diagnosis and prevention of BNoV. Specifically, existing technologies have the following shortcomings: 1. Inefficiency of traditional methods: Due to the difficulty in culturing BNoV in vitro and the lack of mature animal infection models, traditional virus isolation, culture, and electron microscopy detection methods are not only time-consuming and labor-intensive, requiring sophisticated equipment, but also have low sensitivity, failing to meet the needs of rapid screening. 2. Insufficient sensitivity of immunological methods: While methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatographic test strips based on antigen-antibody reactions are relatively simple to operate, their detection sensitivity is low, prone to false negative results, and difficult to effectively distinguish between different viral genotypes. 3. Dependence on specialized equipment for molecular detection methods: Although conventional RT-PCR and real-time quantitative PCR (qPCR) have high sensitivity and specificity, the detection process relies on sophisticated thermal cycling instruments and specialized technicians, resulting in long detection cycles and high costs, making it difficult to promote and apply in grassroots or field environments. 4. Existing isothermal amplification techniques have defects: Although recombinase-mediated isothermal amplification techniques (such as RAA) are fast and have low equipment requirements, their primer design is complex, and aerosol contamination is easily generated when the amplified product is opened for detection, leading to false positive results.

[0004] In recent years, CRISPR-Cas systems have shown great potential in the field of nucleic acid detection due to their high sensitivity and specificity. Among them, the CRISPR-Cas12a system can activate its non-specific trans-cleavage activity after recognizing target nucleic acids, thereby cleaving reporter molecules to generate a signal. However, there are currently no reports of successfully combining reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR-Cas12a technology for rapid on-site detection of BNoV. Therefore, developing a BNoV detection method that combines the convenience of isothermal amplification with the high specificity of CRISPR detection is of significant practical importance for the prevention and control of bovine diarrhea. Summary of the Invention

[0005] Therefore, to address the shortcomings and deficiencies of existing early detection and control technologies for bovine norovirus (BNoV) infection, this invention aims to provide a rapid, on-site BNoV detection method that is less instrument-dependent, quick and simple to operate, highly sensitive, and specific. This method is of great significance for the early detection, diagnosis, and control of bovine norovirus infection. This invention provides primer combinations, kits, and applications for detecting bovine norovirus based on RTRPA-CRISPRCas12a. The specific technical solution is as follows: A crRNA for detecting bovine norovirus, said crRNA having a nucleotide sequence as shown in any of SEQ ID NO. 1-8.

[0006] Furthermore, the application of the described crRNA in the preparation of bovine norovirus detection products.

[0007] In addition, the present invention provides a kit for the accurate and rapid detection of bovine norovirus based on CRISPR / Cas12a technology, wherein the kit contains the crRNA as described in claim 1.

[0008] Furthermore, the kit also contains RT-RPA amplification primer pairs for amplifying bovine norovirus target nucleic acids, the sequences of which are shown in SEQ ID NO.10-19.

[0009] Furthermore, the kit also contains a single-stranded DNA fluorescent reporter molecule with the sequence 5′-FAM-TTATT-BHQ1-3′.

[0010] Furthermore, the kit also includes RT-RPA amplification system reagents and CRISPR / Cas12a detection system reagents.

[0011] In addition, the present invention also provides a CRISPR / Cas12a system for detecting bovine norovirus, the system comprising: (a) The crRNA described above; (b) Cas12a protein; (c) Single-stranded DNA reporter molecules containing fluorescently labeled groups; (d) RT-RPA amplification primer pair used to amplify bovine norovirus target nucleic acid.

[0012] Further, the sequences of the RT-RPA amplification primer pair are shown in SEQ ID NO.10-19; and / or, the sequence of the single-stranded DNA reporter molecule is 5′-FAM-TTATT-BHQ1-3′.

[0013] In addition, the present invention also provides a method for accurate and rapid detection of bovine norovirus based on RT-RPA-CRISPR / Cas12a technology, the method comprising the following steps: (1) Extract nucleic acid from the sample to be tested; (2) Using the nucleic acid extracted in step (1) as a template, reverse transcription recombinase polymerase amplification reaction was carried out using RT-RPA amplification primer pair to obtain the amplification product; (3) The amplification product obtained in step (2) is mixed with a CRISPR / Cas12a detection system containing Cas12a protein, the crRNA and single-stranded DNA reporter molecule, and reacted. (4) Detect the fluorescence signal of the reaction system and determine whether the sample to be tested contains bovine norovirus based on the fluorescence signal.

[0014] Further, the sequences of the RT-RPA amplification primer pair are shown in SEQ ID NO.10-19; and / or, the sequence of the single-stranded DNA reporter molecule is 5′-FAM-TTATT-BHQ1-3′.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention combines reverse transcription recombinase polymerase amplification (RT-RPA) technology with the CRISPR / Cas12a system, and has been successfully applied to the detection of bovine norovirus (BNoV). This method integrates the high efficiency of isothermal amplification with the ultra-high specificity of the CRISPR / Cas system, constructing a novel BNoV nucleic acid detection technology system, and providing a practical new solution for rapid on-site diagnosis of BNoV.

[0016] 2. This invention significantly improves the detection limit by screening the optimal combination of crRNA and RPA primers. Experimental results show (see...) Figure 3 , Figure 4 The method described in this invention has a detection limit as low as 50 copies / reaction, and its sensitivity is more than 10 times higher than that of the traditional quantitative PCR (qPCR) method. This high sensitivity enables it to effectively detect weakly positive samples with extremely low viral loads, extending the detection window period and playing an important role in the early monitoring of BNoV infection.

[0017] 3. The core of this invention lies in the design of crRNA (such as crRNA-2 shown in SEQ ID NO. 2) targeting the conserved and specific target region of BNoV. With excellent specificity, the method of this invention can accurately identify BNoV, and shows no cross-reaction with common bovine pathogens such as bovine coronavirus (BCoV), bovine rotavirus (BRV), and bovine viral diarrhea virus (BVDV), effectively avoiding false positives and ensuring the accuracy of the test results.

[0018] 4. The detection method established in this invention does not rely on expensive thermal cycling equipment (such as real-time quantitative PCR instruments), but only requires a constant temperature incubator or water bath (40℃) and a portable fluorescence detector. The entire detection process (from RT-RPA amplification to CRISPR detection) can be completed within 30-60 minutes, far shorter than the 1.5-2 hours of traditional qPCR methods. This characteristic frees the detection from the limitations of professional laboratories, making it ideal for rapid on-site screening in resource-constrained settings such as farms and fields.

[0019] 5. By optimizing the core components, crRNA and primers, this invention can efficiently and specifically amplify the BNoV target region, resulting in excellent overall detection performance.

[0020] 6. This invention eliminates the need for expensive large-scale equipment such as real-time quantitative PCR instruments, and the cost of reagents and consumables is controllable. The cost per test of the method described in this invention is significantly lower than that of traditional qPCR methods. Furthermore, the operation process is simple and can be completed by professionals without rigorous training, which facilitates its widespread application in grassroots veterinary stations and large-scale cattle farms. It has significant practical value for the daily monitoring, clinical diagnosis, and comprehensive prevention and control of BNoV. Attached Figure Description

[0021] The invention will be further understood from the following description taken in conjunction with the accompanying drawings. The components in the drawings are not necessarily drawn to scale, but rather the emphasis is on illustrating the principles of the embodiments. In different views, the same reference numerals designate corresponding parts.

[0022] Figure 1 The results of fluorescence value determination by CRISPR / Cas12a for 30 min during the screening of 8 crRNAs; Figure 2The image shows the agarose gel electrophoresis results of the RT-RPA amplification primer pair after 30 min of amplification. Figure 3 Fluorescence curve analysis results of CRISPR / Cas12a detection of BNoV sensitivity after serial dilution of BNoV RNA target standard; Figure 4 After serial dilution of BNoV RNA standards, fluorescence signals were detected using CRISPR / Cas12a to detect BNoV sensitivity. Figure 5 Fluorescence curve analysis results for CRISPR / Cas12a detection specificity analysis; Figure 6 Final fluorescence bar chart analysis results of CRISPR / Cas12a detection specificity assay; Figure 7 The results of fluorescence curve analysis of the CRISPR / Cas12a detection method for 20 samples; Figure 8 The results of the final fluorescence histogram analysis of the CRISPR / Cas12a detection method for 20 samples are shown. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to its embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the scope of protection of the invention.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0025] One embodiment of the present invention provides a crRNA for detecting bovine norovirus, wherein the nucleotide sequence of the crRNA is shown in any of SEQ ID NO. 1-8.

[0026] In one embodiment, the crRNA is used in the preparation of bovine norovirus detection products.

[0027] In addition, the present invention provides a kit for the accurate and rapid detection of bovine norovirus based on CRISPR / Cas12a technology, wherein the kit contains the crRNA as described in claim 1.

[0028] In one embodiment, the kit further comprises an RT-RPA amplification primer pair for amplifying bovine norovirus target nucleic acid, the sequence of which is shown in SEQ ID NO.10-19.

[0029] In one embodiment, the kit further comprises a single-stranded DNA fluorescent reporter molecule with the sequence 5′-FAM-TTATT-BHQ1-3′.

[0030] In one embodiment, the kit further comprises individually packaged RT-RPA amplification system reagents and CRISPR / Cas12a detection system reagents.

[0031] In addition, the present invention also provides a CRISPR / Cas12a system for detecting bovine norovirus, the system comprising: (a) The crRNA; (b) Cas12a protein; (c) Single-stranded DNA reporter molecules containing fluorescently labeled groups; and (d) RT-RPA amplification primer pair used to amplify bovine norovirus target nucleic acid.

[0032] In one embodiment, the sequence of the RT-RPA amplification primer pair is shown in SEQ ID NO.10-19; and / or, the sequence of the single-stranded DNA reporter molecule is 5′-FAM-TTATT-BHQ1-3′.

[0033] In addition, the present invention also provides a method for accurate and rapid detection of bovine norovirus based on RT-RPA-CRISPR / Cas12a technology, the method comprising the following steps: (1) Extract nucleic acid from the sample to be tested; (2) Using the nucleic acid extracted in step (1) as a template, reverse transcription recombinase polymerase amplification reaction was carried out using RT-RPA amplification primer pair to obtain the amplification product; (3) The amplification product obtained in step (2) is mixed with a CRISPR / Cas12a detection system containing Cas12a protein, the crRNA and a single-stranded DNA reporter molecule, and reacted. (4) Detect the fluorescence signal of the reaction system and determine whether the sample to be tested contains bovine norovirus based on the fluorescence signal.

[0034] In one embodiment, the sequence of the RT-RPA amplification primer pair is shown in SEQ ID NO.10-19; and / or, the sequence of the single-stranded DNA reporter molecule is 5′-FAM-TTATT-BHQ1-3′.

[0035] Selecting a suitable target region involves designing and screening RPA primers capable of efficiently amplifying that region. Next, the PAM sequence needs to be located within this region, allowing for the screening of suitable crRNAs within the target region. These crRNAs are then combined with highly efficient RPA primers to screen for highly efficient crRNAs, ultimately establishing a CRISPR / Cas detection method for BNoV. A suitable target is one of the key steps in determining detection specificity. The BNoV genome sequence is analyzed, and regions with high intraspecific conservation and interspecific specificity are selected through BLAST and MEGA software alignment analysis. RPA primers and crRNAs are designed within these regions, and the most efficient combinations are screened by combining them.

[0036] This invention reveals that when bovine norovirus nucleic acid forms a ternary complex with Cas12a and crRNA, the DNase activity associated with Cas12a in this complex is activated, cleaving fluorescently labeled single-stranded DNA (ssDNA reporter). The presence of bovine norovirus nucleic acid in the sample can be determined by detecting the fluorescence. Based on this, this invention constructs a rapid and accurate method for detecting bovine norovirus using CRISPR / Cas12a. A high-quality RPA primer and crRNA combination with the highest accuracy, efficiency, and sensitivity was designed to ensure effective bovine norovirus detection.

[0037] The embodiments of the present invention will be described in detail below with reference to specific examples. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0038] Example 1: crRNA design: The core of the CRISPR / Cas12a detection method lies in crRNA; therefore, the quality of crRNA is directly related to the sensitivity and accuracy of the detection method. To screen for the most efficient crRNA, we synthesized target standard RNA for BNoV detection based on the BNoV gene sequence and prepared eight crRNAs (sequences shown in SEQ ID NO. 1-8).

[0039] The template for the crRNA DR sequence was synthesized using two complementary primers, and the annealing system is shown in Table 1.

[0040] Table 1: Annealing System

[0041] Add the components from Table 1 above to a 200 μl centrifuge tube, mix well, and centrifuge. Place the tube in a PCR instrument for annealing: pre-denaturation at 95℃ for 1 min; denaturation at 95℃ for 10 s, annealing at 55℃ for 20 s, extension at 72℃ for 4 s, repeat for 33 cycles; final extension at 72℃ for 3 min; obtain a double-stranded DNA template containing the T7 promoter for crRNA. Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0042] The DNA template was purified by nucleic acid coprecipitation, and the concentration was determined on a Qubit 3.0. The purified DNA template was stored at -20°C.

[0043] crRNA was transcribed in vitro using the Full Gold T7 transcription kit. The transcription system is shown in Table 2.

[0044] Table 2: Transcription System

[0045] Add the components of the transcription system in Table 1 above to a 1.5 mL centrifuge tube, gently mix, and centrifuge. Incubate overnight at 37°C; then add 30 μL of DEPC water and 1.5 μL of DNase I, and incubate at 37°C for 30 min to obtain crRNA.

[0046] RNA was purified using the Trizol method, and the concentration was determined on a Qubit 3.0. The purified crRNA was stored at -80°C.

[0047] Example 2: Synthesis of RNA standards: To establish a CRISPR / Cas12a detection method for BNoV, this invention selects the target region for detection based on the reference sequence Newbury2 through specificity and conservation analysis. We synthesized a target standard RNA for BNoV detection, the sequence of which is shown in SEQ ID NO. 9.

[0048] Using the synthesized plasmid as a template and universal primers, the target double-stranded DNA template was amplified by PCR. The annealing system is shown in Table 3.

[0049] Table 3: Annealing System

[0050] Add the components of the annealing system in Table 3 above to a 200 μl centrifuge tube, mix well and centrifuge. Place in a PCR instrument for annealing reaction: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 20 s, 72℃ extension for 63 s, repeat for 33 cycles, and finally extend at 72℃ for 5 min; obtain the DNA template of the RNA standard sequence. Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0051] The target RNA standard was transcribed in vitro using the T7 transcribing kit. The transcription system is shown in Table 4.

[0052] Table 4: Transcription System

[0053] Add the components of the transcription system from Table 4 above to a 1.5 mL centrifuge tube, gently mix, and centrifuge. Incubate at 37°C for 12 h; then add 2 μL DNase I and 40 μL DEPC H2O, and incubate at 37°C for 30 min to obtain RNA.

[0054] RNA was purified using the Trizol method, and the concentration was determined on a Qubit 3.0. The purified target RNA standard was stored at -80°C.

[0055] Example 3: The most efficient crRNA for screening: Using synthesized target RNA standards and crRNAs, the most efficient crRNAs were screened. The eight crRNAs prepared above were used in the reaction system, with a reaction time of 30 min. Real-time fluorescence values ​​were read using a Roche Light Cycler 480 real-time PCR instrument. The annealing system is shown in Table 5.

[0056] Table 5: Annealing System

[0057] The results are as follows Figure 1 As shown, the crRNA-2 fluorescence curve was the highest at a reaction time of 30 min, indicating that among the eight crRNAs, this crRNA had the best effect and the highest sensitivity.

[0058] Example 4: Establishment of a CRISPR / Cas12a method for detecting BNoV: RT-RPA amplification: The RPA-F / RPA-R sequences designed for RPA amplification are shown in SEQ ID NO.10-19. The RPA amplification systems are shown in Table 6.

[0059] Table 6: RPA amplification system

[0060] The premixed system was prepared using the RNA Isothermal Rapid Amplification Kit (Liquid Basic Type). After thorough mixing, the reaction tubes were incubated at 40°C for 30 min to complete RT-RPA amplification. The RPA amplification products were subjected to 2% agarose gel electrophoresis to observe the band positions and brightness. The RNA Isothermal Rapid Amplification Kit (Liquid Basic Type) was purchased from AmpMed.

[0061] CRISPR / Cas12a detection of BNoV, CRISPR / Cas12a detection system (Table 7).

[0062] Table 7: CRISPR / Cas12a detection system

[0063] The components listed in Table 7 were added sequentially to 8-tube sets. After centrifugation, the tubes were placed in a Roche Light Cycler 480 real-time PCR instrument. The FAM channel was selected, and the reaction was allowed to proceed for 30 minutes before exporting the fluorescence data. The synthesized FAM-BHQ reporter sequence was 5′-FAM-TTATT-BHQ1-3′. The detection conditions were: cap temperature 45℃, well temperature 40℃, reaction time 30 minutes, and fluorescence readings every 1 minute.

[0064] Example 5: CRISPR / Cas12a Sensitivity Analysis for BNoV Detection: RNA positive standards with known viral copy numbers were serially diluted 10-fold to obtain 0 / 10 / 50 / 100 / 1000 copies / 10000 copies. RT-RPA amplification was performed according to the RT-RPA amplification method and conditions in Example 4. Then, the reaction was carried out according to the CRISPR / Cas12a detection method and conditions in Example 4. The RT-RPA templates in the reaction system were the RNA positive standards diluted above with different copies. The fluorescence values ​​were read using a Roche Light Cycler 480 real-time PCR instrument.

[0065] Example 6: CRISPR / Cas12a detection of BNoV specificity analysis: Experimental materials: Synthetic RNA standards for BCoV, BRV, and BVDV.

[0066] RNA standards for BCoV, BRV, and BVDV were synthesized using the BNoV RNA standard synthesis method described in Example 2. Then, BNoV specificity analysis was performed according to the CRISPR / Cas12a detection method described in Example 3, and fluorescence values ​​were read using a Roche Light Cycler 480 real-time PCR instrument.

[0067] The results are as follows Figure 5 , Figure 6 As shown, BCoV, BRV, and BVDV did not fluoresce, while the BNoV sample exhibited a higher fluorescence value. These results demonstrate that the BNoV detection method constructed in this invention has strong specificity and shows no cross-reactivity with other viruses.

[0068] Example 7: CRISPR / Cas12a testing of clinical samples.

[0069] Using the crRNA-2 and the detection method of Example 3, 20 clinical samples were tested (including 3 BNoV GIII.1 genotype positive samples, 3 BNoV GIII.2 genotype positive samples, and 14 negative samples).

[0070] The results are as follows Figure 7 , Figure 8 As shown in the fluorescence curve, the CRISPR / Cas12a detection method has high sensitivity.

[0071] The nucleotide sequence of the crRNA is shown in SEQ ID NO.1-8, and for details, please refer to the nucleotide sequence listing.

[0072] Table 8: Nucleotide sequence of crRNA

[0073] BNoV RNA targets: SEQ ID NO.9:

[0074] Table 9: RPA amplification primers

[0075] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0076] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A crRNA for detecting bovine norovirus, characterized in that, The nucleotide sequence of the crRNA is shown in any of SEQ ID NO. 1-8.

2. The application of the crRNA as described in claim 1 in the preparation of bovine norovirus detection products.

3. A reagent kit for the accurate and rapid detection of bovine norovirus based on CRISPR / Cas12a technology, characterized in that, The kit contains the crRNA as described in claim 1.

4. The reagent kit according to claim 3, characterized in that, The kit also contains RT-RPA amplification primer pairs for amplifying bovine norovirus target nucleic acids, the sequences of which are shown in SEQ ID NO.10-19.

5. The reagent kit according to claim 4, characterized in that, The kit also contains a single-stranded DNA fluorescent reporter molecule with the sequence 5′-FAM-TTATT-BHQ1-3′.

6. The reagent kit according to claim 5, characterized in that, The kit also includes RT-RPA amplification system reagents and CRISPR / Cas12a detection system reagents.

7. A CRISPR / Cas12a system for detecting bovine norovirus, characterized in that, The system includes: (a) The crRNA as described in claim 1; (b) Cas12a protein; (c) Single-stranded DNA reporter molecules containing fluorescently labeled groups; (d) RT-RPA amplification primer pair used to amplify bovine norovirus target nucleic acid.

8. The CRISPR / Cas12a system according to claim 7, characterized in that, The sequences of the RT-RPA amplification primer pairs are shown in SEQ ID NO.10-19; and / or, the sequence of the single-stranded DNA reporter molecule is 5′-FAM-TTATT-BHQ1-3′.

9. A method for accurate and rapid detection of bovine norovirus based on RT-RPA-CRISPR / Cas12a technology, characterized in that, The method includes the following steps: (1) Extract nucleic acid from the sample to be tested; (2) Using the nucleic acid extracted in step (1) as a template, reverse transcription recombinase polymerase amplification reaction was carried out using RT-RPA amplification primer pair to obtain the amplification product; (3) The amplification product obtained in step (2) is mixed with a CRISPR / Cas12a detection system containing Cas12a protein, the crRNA described in claim 1 and a single-stranded DNA reporter molecule, and reacted. (4) Detect the fluorescence signal of the reaction system and determine whether the sample to be tested contains bovine norovirus based on the fluorescence signal.

10. The method according to claim 9, characterized in that, The sequences of the RT-RPA amplification primer pairs are shown in SEQ ID NO. 10-19; and / or, the sequence of the single-stranded DNA reporter molecule is 5′-FAM-TTATT-BHQ1-3′.

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