Primer-based detection
Label one primer with Biotin and the other primer with Fluorescein (FI) (see Figure A.1). The amplicon is detected on the T line (test line), while the C line serves as a control for the flow function.

Probe-based detection
Label one the two primers with Biotin and the detection probe with Fluorescein (FI). Make sure that the probe is complementary to the DNA strand extended from the biotin-labeled primer (see Figure A.2).

To generate biotin-labeled single-stranded amplicon for probe hybridization, asymmetric HDA or PCR should be performed. The unlabeled primer to biotin labeled primer ratio should range between 1:2 and 1:4. Generally, the detection probe concentration needs to be approximately 50 nM. Biotin/FI dual labelled amplicon is detected on T line of the strip.

Type II BESt™ Cassette: two amplicon detection

The Type II cassette is designed to detect two amplicons with two distinct probes, for example, one for the target of interest and one for an internal control. To generate biotin-labeled single-stranded amplicon for probe hybridization, asymmetric HDA or PCR should be performed.

Amplicon 1
Label one of the two primers with Biotin and the detection probe with Digoxgenin. Design the probe so that it is complementary to the DNA strand extended from Biotin-labeled primer (see Figure B). This amplicon is detected on the C line.

Amplicon 2
Label one of the two primers with biotin and the detection probe with Fluorescein (FI). Design the probe so that it is complementary to the DNA strand extended from Biotin-labeled primer (see Figure B). This amplicon is detected on the T line (test line).

Caution:
Since the amplicon 2 with Fluorescein (FI) is detected first by color-particles based on its closer to the flow of colored particles, the T line for FI-amplicon is stronger than the C line when the amount of amplicon 1 is equal to amplicon 2. To detect both amplicons simultaneously with similar intensity of T and C lines, amplification reaction needs to be adjusted so that more amplicon 1(DIG) is produced than amplicon 2 (FI).