Primer-based labeling
Both primers for amplification are labeled: one primer with Biotin and the other primer with FI or DIG.

Probe-based labeling
Only one of two primers is label with Biotin, and a detection probe is labeled with FI or DIG. The detection probe should be complementary to the DNA strand extended from the biotin-labeled primer.

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.

Amplicon Detection

Biotin/FI dual labelled amplicon is detected on T line of the strip, and Biotin/DIG dual-labeled amplicon is detected on C line of the strip.

Detection on C line
C line on the detection strip contains anti-DIG antibody, and detects Biotin/DIG dual-labeled amplicon.

Detection on T line
T line on the detection strip contains anti-FI antibody, and detects Biotin/FI dual-labeled amplicon.

Caution:
When the amounts of two amplicon are equal, T line is stronger than C line, since T line is closer to the flow of amplicons than C line is. To detect both amplicons simultaneously with similar intensity of T and C lines, amplification reaction needs to be adjusted so that more Biotin/DIG amplicon is produced than Biotin/FI amplicon.