Our project aims to develop a rapid, sensitive, and straightforward detection method for identifying the single nucleotide variant C677T in the MTHFR gene. By utilizing recombinant special probes, RNase H II, and a PCR amplification system, this approach enables the identification of CC, CT, or TT genotypes in a one-tube reaction, facilitating early detection of risks associated with folate metabolism disorders, thereby contributing to the health of pregnant women and their fetuses.
To achieve this goal, after completing principle verification and performance optimization based on the plasmid, we applied this method to the detection of actual blood samples to test its accuracy and precision. The test results demonstrate that this method's genotype identification for blood samples is completely consistent with the gold standard sequencing results, achieving 100% accuracy. Multiple repeated experiments conducted on the same sample yielded consistent results, indicating excellent precision of the proposed approach.
In addition, since we used a qPCR instrument to detect fluorescent signals, we conducted extensive testing on the device, including inter-day precision, intra-day precision, repeatability, and sensitivity validation. The tests demonstrated that the instrument we used performs excellently.
Introduction
Contributions and Achievements in Measurement
(1) Our project can determine the specific genotypes (CC, CT, and TT) of the MTHFR 677 single nucleotide polymorphism through a single-tube reaction, which is easy to perform, time-efficient, and cost-effective.
(2) The experimental method we developed has been successfully applied to the detection of blood samples, with genetic typing results confirmed through comparison with sequencing data. The project demonstrated an accuracy of 100%, along with excellent precision.
(3) Compared to existing detection techniques, our technology significantly simplifies experiments, including both experimental design and operational procedures. The TaqMan approach heavily relies on probe length and Tm values, which are determined by the target sequence. However, target gene sequences vary widely, with bases distributed randomly. Certain genes consistently fail to achieve suitable Tm values (e.g., fragments with high poly T or poly A content), making it difficult to design TaqMan-compatible probes. Our method completely eliminates this limitation. By replacing conventional temperature-controlled recognition with the specific recognition and cleavage of RNase H II, it enables highly flexible probe design. In addition, we only need to perform a simple blood pre-treatment, then place the sample into a qPCR machine for reaction and result output. According to the protocol, the experiment can be fully replicated without requiring extensive professional training.
(4) By developing algorithmic modules that process qPCR fluorescence data, our system analyzes and statistically processes the information. Using predefined genotype models, it accurately identifies genetic profiles and generates standardized reports. This automated solution dramatically lowers operational barriers with a user-friendly interface. Operators need only follow step-by-step instructions to obtain precise results without requiring specialized training. More importantly, it eliminates time-consuming manual identification processes and reduces training costs for professionals. The system also prevents diagnostic errors caused by human error, significantly lowering overall expenses.
(2) The experimental method we developed has been successfully applied to the detection of blood samples, with genetic typing results confirmed through comparison with sequencing data. The project demonstrated an accuracy of 100%, along with excellent precision.
(3) Compared to existing detection techniques, our technology significantly simplifies experiments, including both experimental design and operational procedures. The TaqMan approach heavily relies on probe length and Tm values, which are determined by the target sequence. However, target gene sequences vary widely, with bases distributed randomly. Certain genes consistently fail to achieve suitable Tm values (e.g., fragments with high poly T or poly A content), making it difficult to design TaqMan-compatible probes. Our method completely eliminates this limitation. By replacing conventional temperature-controlled recognition with the specific recognition and cleavage of RNase H II, it enables highly flexible probe design. In addition, we only need to perform a simple blood pre-treatment, then place the sample into a qPCR machine for reaction and result output. According to the protocol, the experiment can be fully replicated without requiring extensive professional training.
(4) By developing algorithmic modules that process qPCR fluorescence data, our system analyzes and statistically processes the information. Using predefined genotype models, it accurately identifies genetic profiles and generates standardized reports. This automated solution dramatically lowers operational barriers with a user-friendly interface. Operators need only follow step-by-step instructions to obtain precise results without requiring specialized training. More importantly, it eliminates time-consuming manual identification processes and reduces training costs for professionals. The system also prevents diagnostic errors caused by human error, significantly lowering overall expenses.
Analysis Performance in Blood Samples
1. Accuracy of Blood Samples
Accuracy refers to the degree to which the results obtained by the established method are close to the true values. The gold standard for determining the precise sequence of genes is to use gene sequencing methods. We use our method to detect blood samples and determine genotypes based on the results, then compare these findings with sequencing data to verify their accuracy.
Experimental Procedure
1. Sample Preparation: Take 100 μL of different blood samples. There are 50 blood samples.
CAUTION: Use aseptic technique during blood procession to prevent contamination.
PAUSE POINT: The tube containing the sample and lysis buffer can be kept on ice for a short period (~30 min) before proceeding to the mixing step if multiple samples are being processed.
2. Lysis: Mix the sample with 500 μL of red blood cell lysis buffer in a centrifuge tube.
CAUTION: Pre-cool the lysis buffer to 4 °C to improve red blood cell lysis efficiency.
3. Mixing: Gently invert the centrifuge tube 10 times to ensure thorough mixing.
CAUTION: Gently invert the tube to avoid vigorous shaking, which may damage white blood cells.
4.Incubation: Place the centrifuge tube in a 4 °C refrigerator and incubate for 20 minutes.
CAUTION: Ensure consistent incubation time and temperature for all samples and replicates to achieve comparable lysis efficiency.
5. Centrifugation: After incubation, centrifuge the sample at 5000 rpm for 5 min.
CAUTION: Before using the centrifuge, ensure that the solution volumes in the centrifuge tubes are equal to prevent instrument damage caused by imbalanced weights.
CRITICAL STEP: This step involves centrifuging the white blood cells to the bottom of the tube, separating them from the lysed red blood cell fragments. Balance tubes before centrifugation.
6. Remove supernatant: Carefully discard the supernatant to avoid disturbing the precipitation, which primarily containing white blood cells.
CRITICAL STEP: Avoid touching the pellet with the pipette tip to prevent loss of cellular material containing the DNA template. It is often better to leave a small amount of supernatant than to risk disturbing the precipitation.
7. Resuspend: Add 50 μL of red blood cell lysis buffer to the precipitate and mix to form a suspension.
CRITICAL STEP: The suspension in this step is mainly used as a DNA template for subsequent reactions. The solution for dissolving white blood cells can also be sterilized deionized water. If white blood cells are dissolved in water, DNA will be immediately released. If dissolved in red blood cell lysate, DNA will be released during PCR heating at 95 °C.
8. qPCR Reaction: Take 5 μL of the suspension and use it directly as the DNA template for the qPCR reaction. Prepare the qPCR reaction mixture with a total volume of 25 μL, as outlined in the table1 below. The nucleic acid sequences are shown in Table 2.
CAUTION: Use aseptic technique during blood procession to prevent contamination.
PAUSE POINT: The tube containing the sample and lysis buffer can be kept on ice for a short period (~30 min) before proceeding to the mixing step if multiple samples are being processed.
2. Lysis: Mix the sample with 500 μL of red blood cell lysis buffer in a centrifuge tube.
CAUTION: Pre-cool the lysis buffer to 4 °C to improve red blood cell lysis efficiency.
3. Mixing: Gently invert the centrifuge tube 10 times to ensure thorough mixing.
CAUTION: Gently invert the tube to avoid vigorous shaking, which may damage white blood cells.
4.Incubation: Place the centrifuge tube in a 4 °C refrigerator and incubate for 20 minutes.
CAUTION: Ensure consistent incubation time and temperature for all samples and replicates to achieve comparable lysis efficiency.
5. Centrifugation: After incubation, centrifuge the sample at 5000 rpm for 5 min.
CAUTION: Before using the centrifuge, ensure that the solution volumes in the centrifuge tubes are equal to prevent instrument damage caused by imbalanced weights.
CRITICAL STEP: This step involves centrifuging the white blood cells to the bottom of the tube, separating them from the lysed red blood cell fragments. Balance tubes before centrifugation.
6. Remove supernatant: Carefully discard the supernatant to avoid disturbing the precipitation, which primarily containing white blood cells.
CRITICAL STEP: Avoid touching the pellet with the pipette tip to prevent loss of cellular material containing the DNA template. It is often better to leave a small amount of supernatant than to risk disturbing the precipitation.
7. Resuspend: Add 50 μL of red blood cell lysis buffer to the precipitate and mix to form a suspension.
CRITICAL STEP: The suspension in this step is mainly used as a DNA template for subsequent reactions. The solution for dissolving white blood cells can also be sterilized deionized water. If white blood cells are dissolved in water, DNA will be immediately released. If dissolved in red blood cell lysate, DNA will be released during PCR heating at 95 °C.
8. qPCR Reaction: Take 5 μL of the suspension and use it directly as the DNA template for the qPCR reaction. Prepare the qPCR reaction mixture with a total volume of 25 μL, as outlined in the table1 below. The nucleic acid sequences are shown in Table 2.
Table 1. The reaction system in one-tube.
Reaction program: 94 ℃,5 min + (94 ℃,30 s + 55 ℃,30 s + 68 ℃,25 s) × 45 cycles
class="bold-text">CAUTION: Special workstations and pipette tips are used to prevent PCR contamination. Keep master mix on ice to maintain enzyme activity. Prevent cross-contamination between samples, especially with components like RNase H II.
Table 2. The relevant nucleic acid sequences.
/rG/ and /rA/ are RNA bases.
9. Process 50 different blood samples following the above steps. Input the experimental data into our team's self-developed program, which determines the genotype (CC, CT or TT) based on the output fluorescence signal, and then compare these results with sequencing data.
Results
According to the determination criteria we have established:
∆∆Rn = ∆Rn (FAM) - ∆Rn (ROX)
If ∆∆Rn > 0, then
Relative Difference = (∆Rn FAM-∆Rn ROX)/ ∆Rn FAM
If ∆∆Rn < 0, then
Relative Difference = (∆Rn ROX-∆Rn FAM)/ ∆Rn ROX
When the ∆∆Rn > 50000 and Relative Difference ranges from 60 to 120%, the sample is classified as CC type. Conversely, if the ∆∆Rn < -50000 and Relative Difference is between 60 and 120%, the sample is classified as TT type. When the ∆∆Rn value falls within the range of 0 to 60%, the sample is classified as CT type.
∆∆Rn = ∆Rn (FAM) - ∆Rn (ROX)
If ∆∆Rn > 0, then
Relative Difference = (∆Rn FAM-∆Rn ROX)/ ∆Rn FAM
If ∆∆Rn < 0, then
Relative Difference = (∆Rn ROX-∆Rn FAM)/ ∆Rn ROX
When the ∆∆Rn > 50000 and Relative Difference ranges from 60 to 120%, the sample is classified as CC type. Conversely, if the ∆∆Rn < -50000 and Relative Difference is between 60 and 120%, the sample is classified as TT type. When the ∆∆Rn value falls within the range of 0 to 60%, the sample is classified as CT type.
Figure 1. The genotype results output by the program. The red dots in the pink color block are TT type, the blue dots in the light blue color block are CC type, and the purple dots in the light purple color block are heterozygous CT.
Figure 2. Partial sequencing results. If the peak corresponding to the base is a single red peak, it indicates a homozygous mutant (TT); if it is a single blue peak, it indicates a homozygous wild-type (CC); if the blue and red peaks overlap, it indicates a heterozygous (CT) genotype.
According to the typing results shown in Figure 1 and the sequencing results presented in Figure 2, the typing of all the samples we analyzed is consistent with the sequencing results, demonstrating an accuracy of 100%.
2. Precision of Blood Samples
Precision refers to the degree of closeness between the results obtained from multiple sampling measurements of the same homogeneous sample under specified conditions, typically expressed as the relative standard deviation (RSD). We selected a blood sample and conducted ten tests following the same experimental protocol. We then analyzed the results to determine whether the precision is excellent.
Experimental Procedure
1. Select a CT-type sample to conduct 10 repeated experiments. The experimental procedures follow the protocol used in the accuracy experiment.
2. Simultaneously monitor both FAM and ROX fluorescence signals, select ∆Rn from the 40th cycle of the amplification curve for each experiment to plot
3. Calculate the Relative Difference based on ∆Rn value from the 40th cycle of the amplification curve.
2. Simultaneously monitor both FAM and ROX fluorescence signals, select ∆Rn from the 40th cycle of the amplification curve for each experiment to plot
3. Calculate the Relative Difference based on ∆Rn value from the 40th cycle of the amplification curve.
Relative Difference = | (∆Rn FAM-∆Rn ROX)/ ∆Rn FAM |
4. Plot the Ct values of each sample.
CAUTION: Cycle threshold (Ct) refers to the number of cycles experienced when the fluorescence signal reaches the set threshold during PCR amplification, reflecting the initial concentration of the target gene in the sample.
5. Calculate the Relative Standard Deviation (RSD) value for ∆Rn, Relative Difference and Ct.
CAUTION: Cycle threshold (Ct) refers to the number of cycles experienced when the fluorescence signal reaches the set threshold during PCR amplification, reflecting the initial concentration of the target gene in the sample.
5. Calculate the Relative Standard Deviation (RSD) value for ∆Rn, Relative Difference and Ct.
Results
Figure 3. The ∆Rn values of the same sample in ten experiments.
Figure 4. The Ct values of the same sample in ten experiments.
Figure 5. The Relative Difference values of the same sample in ten experiments.
Figure 6. The RSD values of the same sample in ten experiments.
According to the experimental results, the ∆Rn values, Ct values, and relative differences across the ten experiments were very consistent. The RSD was calculated for each parameter, and all RSD values were found to be within the range of 0–10%, indicating excellent experimental precision. In addition, both wild-type and mutant probes in heterozygous blood samples all break and produce corresponding fluorescent signals (FAM and ROX). The fluorescence signals of these two types are very similar, which aligns with the optimized conditions presented in our Engineering and Results studies.
As demonstrated above, our solution exhibits excellent precision.
As demonstrated above, our solution exhibits excellent precision.
3. Intra-day Precision
Intra-day precision refers to the consistency of measurements obtained from repeated analyses of the same sample within a single day.
Experimental Procedure
Take a CT-type blood sample as the test specimen and analyze it three times at different time periods within one day. The experimental procedures follow the protocol used in the accuracy experiment.
Results
Figure 7. The ∆Rn values of the same sample in three experiments conducted on a single day.
Figure 8. The Ct values of the same sample in three experiments conducted on a single day.
Figure 9. The Relative Difference values of the same sample in three experiments conducted on a single day.
Figure 10. The RSD values of the same sample in three experiments conducted on a single day.
According to the experimental results, the ∆Rn values, Ct values, and relative differences across the three experiments were very consistent. The RSD was calculated for each parameter, and all RSD values were found to be within the range of 0–10%, indicating a very low degree of data dispersion relative to the mean.
The consistent measurement results obtained from repeated analyses of the same sample within a single day demonstrate excellent intra-day accuracy.
The consistent measurement results obtained from repeated analyses of the same sample within a single day demonstrate excellent intra-day accuracy.
4. Inter-day precision
Inter-day precision characterizes the consistency of repeated measurements of the same sample taken at different times, reflecting the stability of the measurement system over time.
Experimental Procedure
Take a CT-type blood sample as the test specimen and analyze it every 5 days for one month. The experimental procedures follow the protocol used in the accuracy experiment.
Results
Figure 11. The ∆Rn values of the same sample were measured every five days over the course of one month.
Figure 12. The Ct values of the same sample were measured every five days over the course of one month.
Figure 13. The Relative Difference values of the same sample were measured every five days over the course of one month.
Figure 14. The RSD values of the same sample were measured every five days over the course of one month.
According to the experimental results, the ∆Rn values, Ct values, and relative differences across the three experiments were very consistent. The RSD was calculated for each parameter, and all RSD values were found to be within the range of 0–10%, indicating a very low degree of data dispersion relative to the mean.
The consistent results obtained from repeated measurements of the same sample every five days over the course of one month demonstrate excellent inter-day accuracy.
5. Reproducibility
Reproducibility refers to the ability of other researchers to obtain the same results as we did under identical conditions and using the same experimental protocol.
Experimental Procedure
Take a heterozygous-type blood sample (CT-type) as the test specimen and have three different operators perform the experimental procedures on the same day.
Results
Figure 15. The ∆Rn values of three different operators conducting experiments on the same sample.
Figure 16. The Ct values of three different operators conducting experiments on the same sample.
Figure 17. The Relative Difference values of three different operators conducting experiments on the same sample.
Figure 18. The RSD values of three different operators conducting experiments on the same sample.
According to the experimental results, the ∆Rn values, Ct values, and relative differences across the three experiments were very consistent. The RSD was calculated for each parameter, and all RSD values were found to be within the range of 0–10%, indicating a very low degree of data dispersion relative to the mean.
The consistent results obtained from different operators of the same sample demonstrate excellent reproducibility.
The consistent results obtained from different operators of the same sample demonstrate excellent reproducibility.
Calibration and Standardization
1. Calibration of qPCR instrument
The qPCR instrument is the core equipment of our project, and its performance accuracy directly affects the reliability of the experimental results. Regular calibration and testing of qPCR instruments are essential.
Temperature calibration
Temperature control is crucial in the qPCR reaction process. Different temperature stages (such as denaturation, annealing, and elongation) require precise control to ensure the normal progress of DNA amplification. Therefore, it is essential to verify the deviation between the set temperature and the actual temperature of the PCR instrument.
Method:
(1) Use a high-precision thermometer, such as a platinum resistance thermometer, as a standard and place it inside or near the heating block of the qPCR instrument.
(2) Set different temperature points (e.g., 95 °C, 60 °C, 72 °C) and record the difference between the temperature displayed by the qPCR instrument and the reading from the standard thermometer.
(3) The error of temperature need to remain within the allowable range (±0.5 °C). Adjust the temperature control system of the qPCR instrument based on these differences until the predetermined accuracy requirements are met.
Considering that this operation requires specialized equipment and professional training, we contact the instrument company every six months to request that they send qualified personnel for instrument temperature testing and calibration.
Method:
(1) Use a high-precision thermometer, such as a platinum resistance thermometer, as a standard and place it inside or near the heating block of the qPCR instrument.
(2) Set different temperature points (e.g., 95 °C, 60 °C, 72 °C) and record the difference between the temperature displayed by the qPCR instrument and the reading from the standard thermometer.
(3) The error of temperature need to remain within the allowable range (±0.5 °C). Adjust the temperature control system of the qPCR instrument based on these differences until the predetermined accuracy requirements are met.
Considering that this operation requires specialized equipment and professional training, we contact the instrument company every six months to request that they send qualified personnel for instrument temperature testing and calibration.
Fluorescence calibration
QPCR monitors the DNA amplification process in real time by detecting changes in fluorescence signals. Therefore, fluorescence signal calibration is essential, which ensures that the fluorescence detection system accurately measures fluorescence intensity, enabling precise calculation of reliable quantitative analysis.
Method:
We repeated fluorescence intensity measurements across 96 wells under identical fluorescence conditions and analyzed the consistency of the results.
Method:
We repeated fluorescence intensity measurements across 96 wells under identical fluorescence conditions and analyzed the consistency of the results.
Result:
Figure 19. Thermogram of 96-well fluorescence intensity.
The fluorescence intensity of each well is very similar, with a calculated RSD of 5%, indicating minimal variation in qPCR fluorescence readings across different wells.
2. Sensitivity Verification and Amplification Efficiency
The detection sensitivity and amplification efficiency of qPCR is a key indicator for evaluating its performance. To verify the amplification efficiency of the qPCR instrument, we purchased a complete set of specialized qPCR reaction detection kits, which used monkeypox pseudovirus F3L as the reaction template.
Experimental Procedure
The concentration of Monkeypox Virus F3L was 4.26×105 copies/μL.
1. Prepare ten-fold dilution series of F3L pseudovirus ranging from 105 to 10 copies/μL.
2. According to the directions of Virus DNA-Direct Taqman qPCR Kit, 10 μL F3L pseudovirus were mixed with an equal volume of Buffer V and followed by incubating at 95 °C for 10 min.
3. After the incubation, the mixture was centrifuged at 5000 rpm for 8 min and the supernatant was our DNA template.
4. Quantitative Polymerase Chain Reaction (qPCR) was implemented. Sequences used were shown in Table 3.
1. Prepare ten-fold dilution series of F3L pseudovirus ranging from 105 to 10 copies/μL.
2. According to the directions of Virus DNA-Direct Taqman qPCR Kit, 10 μL F3L pseudovirus were mixed with an equal volume of Buffer V and followed by incubating at 95 °C for 10 min.
3. After the incubation, the mixture was centrifuged at 5000 rpm for 8 min and the supernatant was our DNA template.
4. Quantitative Polymerase Chain Reaction (qPCR) was implemented. Sequences used were shown in Table 3.
Table 3. The relevant nucleic acid sequences in sensitivity verification.
5. Reactions were performed using a 20 μL reaction mixture containing 10 μL 2× TaqMan qPCR Direct Mix, 200 nM each of forward primer and reverse primer, 200 nM of probe, and 4 μL DNA template. All qPCR was analyzed using a QuantStudio 3 Real-Time PCR instrument with thermocycling conditions as follows: 95°C for 3 minutes and 40 cycles of 95 °C for 15 seconds followed by 56 °C for 15 seconds and 72 °C for 30 seconds. The fluorescence signal monitoring pathway is FAM.
Results
Figure 20. The real-time fluorescence curve of different concentration of Monkeypox Virus F3L.
Figure 21. The Ct value forms a linear relationship with the concentration of monkeypox virus F3L.
When the pseudo virus concentration ranged from 10^1 to 10^5, its Ct values formed a standard curve with the equation Y = -3.2271X + 36.844 and R² = 0.9988. Calculating the amplification efficiency using the formula E = [10^(-1/slope) - 1] * 100%, we obtained an amplification efficiency of 103% (amplification efficiencies between 90% and 110% are acceptable). This indicates that the qPCR instrument exhibits good amplification efficiency.
Wide applicability
To demonstrate the broad applicability of our approach, we tried to apply this scheme to the detection of BRAF V600E. The BRAF gene is a proto-oncogene located on the long arm of human chromosome 7 (7q34). V600E mutation is the most common SNP mutation in the BRAF gene, which is associated with thyroid cancer and melanoma. It results from a substitution of the thymine (T) base with adenine (A) at position 1799, leading to the replacement of valine with glutamic acid at amino acid position 600. We designed specific probes based on the corresponding gene sequences, used a plasmid containing a 493 bp BRAF fragment as a template, and followed the MTHFR experimental protocol to detect the BRAF V600E.
Experimental Procedure
Table 3. The relevant nucleic acid sequences for BRAF.
/rA/ and /rU/ are RNA bases.
Results
Figure 22. Real-time fluorescent signal generated by the mutant-type template of BRAF.
Figure 23. Real-time fluorescent signal generated by the wild-type template of BRAF.
Figure 24. Real-time fluorescent signal generated by the heterozygous-type template of BRAF.
The experimental results demonstrated that the output of the qPCR instrument was consistent with the expected outcomes: when only the wild-type template was present, only the fluorescence signal from the wild-type probe showed a significant increase; when only the mutant template was present, only the fluorescence signal from the mutant probe showed a significant increase; and when both wild-type and mutant templates were present in the system, the fluorescence signals from both corresponding probes increased significantly. These findings confirm the feasibility of the detection method for BRAF V600E.
Experimental results demonstrate that the SNP detection method we developed can adjust detection sites according to different testing requirements, exhibiting extremely broad application prospects.
Experimental results demonstrate that the SNP detection method we developed can adjust detection sites according to different testing requirements, exhibiting extremely broad application prospects.
Measurement Award Considerations
Our project has been meticulously designed and documented to meet the standards of the iGEM Measurement Award, which emphasizes ensuring our experimental protocols are reproducible, reliable, and useful to the broader synthetic biology community. Below are key aspects of our project reflecting these guidelines:
(1) Accuracy: We use our method to detect blood samples and determine genotypes based on the results, then compare these findings with sequencing data to verify the accuracy. The experimental results show that the accuracy of our project is as high as 100%.
(2) Precision: We selected a blood sample and conducted ten tests following the same experimental protocol. The experimental results were consistent.
(3) Reproducibility: We ensured reproducibility of each experimental step through multiple trials and three-fold replication, minimizing variation across different experiments. Different experimenters have used our protocol to test the same sample, and the experimental results were all consistent, demonstrating excellent reproducibility.
(4) Controls and Calibration: We ensure the excellent performance of our core instrument qPCR through temperature calibration, fluorescence calibration, and amplification efficiency calculation, reducing the possibility of errors.
(5) Detailed Protocol: Our method for genetic testing via blood samples, including the sensitivity and specificity of the reaction system, blood sample experiments, and RPA experimental protocols, is clearly outlined. These protocols can be easily followed by other iGEM teams, ensuring our measurements are reproducible and verifiable.
(6) Applicability to Other Projects: Our experimental methodology can be applied to other genes, such as BRAF V600E. We are currently utilizing this approach for diagnostics involving other genes.
(1) Accuracy: We use our method to detect blood samples and determine genotypes based on the results, then compare these findings with sequencing data to verify the accuracy. The experimental results show that the accuracy of our project is as high as 100%.
(2) Precision: We selected a blood sample and conducted ten tests following the same experimental protocol. The experimental results were consistent.
(3) Reproducibility: We ensured reproducibility of each experimental step through multiple trials and three-fold replication, minimizing variation across different experiments. Different experimenters have used our protocol to test the same sample, and the experimental results were all consistent, demonstrating excellent reproducibility.
(4) Controls and Calibration: We ensure the excellent performance of our core instrument qPCR through temperature calibration, fluorescence calibration, and amplification efficiency calculation, reducing the possibility of errors.
(5) Detailed Protocol: Our method for genetic testing via blood samples, including the sensitivity and specificity of the reaction system, blood sample experiments, and RPA experimental protocols, is clearly outlined. These protocols can be easily followed by other iGEM teams, ensuring our measurements are reproducible and verifiable.
(6) Applicability to Other Projects: Our experimental methodology can be applied to other genes, such as BRAF V600E. We are currently utilizing this approach for diagnostics involving other genes.
