A novel rapid detection method for a single-nucleotide substitution mutation derived from canine urothelial and prostatic carcinoma cells present in small amounts in urine sediments

Abstract

For early detection of canine urothelial and prostatic carcinoma, we intend to develop and commercialize a simple and rapid detection method for the BRAF V595E mutation, a known mutation in this cancer. Detection of the single-nucleotide substitution in cancer cells contained in urine sediments is effective for early cancer diagnosis. However, urine sediment also contains many normal cells, and when there is a small relative composition of cancer cells, the mutation is difficult to detect by conventional methods other than next-generation sequencing. Our new detection method enables reliable discrimination with the same labor and cost as the PCR method. We compared the results of our new method with the results of the conventional Sanger method for 38 canine urine sediment samples, and the results of 34 samples were consistent between both methods. The remaining four results were all determined to be negative by the Sanger method and positive by our new method. For these four samples, the ratio of the mutated gene to the wild-type gene was estimated using a third-generation sequencer, and the ratio of the mutated gene was 0.1%-1.4%. We postulate that the Sanger method gave a negative result because of the low abundance of the mutated gene in these samples, proving the high sensitivity of our new method.

Fig 1. Detection of the single-nucleotide mutation by PCR with a blocker.

Fig 2. Screening of primers and blockers (a case for reverse primer R8).

Ct values (A) and melting curves (B-D) of the amplified products are shown for all nine combinations with reverse primer R8. The three columns on the left of A show the Ct values with forward primer F4 and blockers BF1, BF2, and BF3 in order from the left. Similarly, the three central columns show those with forward primer F5, and the three right columns show those with F6. Data for Mu as the template are indicated by red circles, for WT by blue circles, for NC1 by black diamonds, and for NC2 by black squares. Markers that did not amplify within 90 cycles are placed outside below the graph area. Markers with melting curve peaks outside the range of 80°C to 81°C, which are judged to be non-specific amplification, are shown in faint colors. Melting curves for Mu as template are shown in B, for NC1 in C, and for WT in D. Melting curves with forward primer F4 are shown by the magenta line, F5 by the yellow-green line, and F6 by the black line, while melting curves with blocker BF1 are shown as a solid line, blocker BF2 as a long-dashed line, and blocker BF3 as a short-dashed line.

Fig 3. Sensitivity test of the three selected detection sets.

The results of the sensitivity test for three combinations of primers and blockers—F4, R6, and BF2 (A and D); F5, R8, and BF2 (B and E); and F4, R8, and BF1 (C and F)—are shown. Two concentrations of the blocker were tested, and the results for 0.4 μM are shown in the upper row (A-C) and for 1.2 μM in the lower row (D-F). The copy numbers of the mutated gene in the test solutions were 9000, 3000, 1000, 333, 111, and 37 in order from the left, and three samples of each copy number were tested. Markers that overlap and are difficult to see are displayed by shifting their positions to the left or right horizontally. Each sample contained 50 ng of salmon DNA and at least 9 × 10³ copies of plasmids containing the wild-type gene. Three samples each of WT and NC1 were tested as the negative controls, and the results are shown on the right line of the graph area. Data that did not amplify within 90 cycles are placed on the underline of the graph area, and markers with melting curve peaks out of the range between 80°C and 81°C are shown in a faint color as they were judged to be non-specific amplification.