Verify the probe effect
The effect of reporter type and reporter concentration on the fluorescence amount was detected by Cas protein. The E gene was digested with Cas protein by various concentrations of the 2020NBT reporter, 2022_5nt reporter and 2022_15nt reporter. From the result，the 2022_15nt reporter had the best effects. After comprehensive consideration of cost and effect, we select 4 μM as actual used concentration.
Figure 1. (A) Fluorescence was detected by UV light
Figure 1. (B)Fluorescence was detected by Microplate Reader.
Verify the primer effect
Hydatid characteristic sequence plasmids were purified after RPA amplification with 16 pairs of primers. The hydatid characteristic sequence plasmids after RPA amplification were purified with phenol and chloroform, and the supernatant was taken after centrifugation for electrophoresis. Based on the results, because there was no band at the expected target fragment, so the two primer pairs, mgs-2 and mgs-8, were removed.
Figure 2. 1.5% agarose gel electrophoresis
Find the lower detection limit
To find the lower detection limit of hydatid DNA in Cas digestion after RPA amplification.
Hydatid characteristic sequence plasmids at different copy numbers were subjected to Cas digestion after RPA amplification and treated with the 2022_15nt probe to detect a lower limit of detection of 10 copies.
Figure 3. Fluorescence was detected by Microplate Reader.
Test the feasibility by single sgRNA treatment
Test the feasibility of detecting the hydatid plasmid with Cas digestion by single sgRNA treatment after RPA amplification with single primers. This indicates that RPA digestion with Cas was effective in detecting hydatid DNA.
Figure 4. (A)Forecast result table
Figure 4. (B)Fluorescence results Table
Figure 4. (C)Table of results of the microplate reader
Test the feasibility by single sgRNA and multiplex sgRNA treatment
Test the feasibility of the hydatid plasmid undergoing Cas digestion by single sgRNA and multiplex sgRNA treatment after RPA amplification with multiple sets of primers. This indicates that the detection of hydatid DNA was effective by multiple primer RPA and Cas digestion with single sgRNA or multiplex sgRNA treatment.
Figure 5. (A)Forecast result table
Figure 5. (B)Fluorescence results Table
Figure 5. (C)Table of results of the microplate reader
Test both positive and negative results with test strips
Test both positive and negative results with test strips, shown as follows,
Positive: C band has a red line and T band has a red line.
Negative: Only C band has a red line, while T band has no red line.
Figure 6. Test strip result diagram
The specificity test
The specificity test of the test method indicates that this test method will not detect other parasitic diseases as the positive, nor detect human DNA as the positive, and the primer sequence is specific to the sgRNA sequence.
Normal human DNA, schistosoma DNA and hydatid DNA were blindly tested, and multiple sgRNA-guided cas12a digestion was performed after multiple primer RPA amplification.
Figure 7. Blind test results of simulated patient samples
Multiple sgRNA can improve detection speed
The necessity of verifying the multiple primer detection method, indicating that the use of Cas digestion detection with multiple sgRNA is more convenient to improve our detection speed.
Simulated patient samples were subjected to Cas digestion with single sgRNA after RPA amplification using single primers.
Figure 8. Single primer review results of simulated patient samples
Figure 9.The comparison of real positive rate between single sequence detection and multiple sequence detection.
