Table 1.
Oligonucleotides used in this study.
Fig 1.
Detection principle of the PCR-LFA.
PCR: A KHV-specific FITC-labeled PCR product is amplified from KHV-DNA. The amplified internal amplification control [IAC] PCR product is double-labeled (DIG and FITC). Hybridization: During post-PCR hybridization, the biotin labeled probe binds to the FITC-labeled strand of the KHV-specific PCR product. This results in a hybridization product which is detectable via LFA. LFA detection principle: The KHV-specific hybridization product is immobilized due to biotin-streptavidin interaction. The IAC PCR product is bound by a stationary anti-digoxigenin [DIG] antibody. The mobile gold-conjugate contains anti-FITC antibodies which specifically bind to the FITC-labeled immobilized PCR products on the pad. Accumulation of gold nanoparticles leads to the appearance of visible test lines. The immunoassay control line [C] is formed due to binding of the mobile anti-FITC antibodies (from goat; gt) by a stationary anti-goat antibody. Appearance of the control line confirms general functionality of the LFA. LFA Interpretation: [+] The test is interpreted as positive if the KHV-specific test line appears. [–] A negative result is defined by absence of the KHV-test line while the IAC-signal is present. [n.v.] If the KHV- and the IAC-signal are absent, the test is interpreted as not valid.
Fig 2.
Test for nonspecific interaction between labeled primers or probes resulting in PCR-independent signals in LFA. The following reaction components were tested: Lane [–] negative control—without primers or probes; lane [1] KHV 163R FITC; lane [2] KHV 109P (rc) BIO; lane [3] IAC-Mix (1x); lane [4] KHV 163R FITC + KHV 109P (rc) BIO; lane [5] KHV 163R FITC + IAC-Mix (1x); lane [6] KHV 109P (rc) BIO + IAC-Mix (1x); lane [7] KHV 163R FITC + KHV 109P (rc) BIO + IAC-Mix (1x); lanes [8–12] 2x KHV-primer mix + KHV 109P (rc) BIO. The red arrow indicates the location where the KHV-specific test line [KHV] would appear. The black arrow indicates the location where the internal amplification control line [IAC] would be seen. Fig 2 was prepared from two raw images, i.e. S3 and S4 Figs.
Fig 3.
Influence of unlabeled helper oligonucleotides on hybridization efficiency.
Two helper oligonucleotides, KHV-Helper 1 (H1) and KHV-Helper 2 (H2), were tested under hybridization conditions. Ten pmol of each helper was added to each hybridization mix including two pmol of the detection probe KHV 109P (rc) BIO. (A) Each helper combination was tested with two KHV-PCR product concentrations (dilution 1:100 and 1:2,000, respectively) in triplicate. Representative test strips are shown: lane 1—without helpers (-H); lane 2—with H1 (+H1); lane 3—with H2 (+H2); lane 4—with H1 and H2 (+H1+H2). [C] immunoassay control line; [KHV] KHV-specific test line indicated by a red arrow. (B) Signal intensities are shown as relative intensities (rel. SI [%]). The intensity of the test line on strip 1 (Fig 3A, lane 1) without helpers was treated as reference. Mean value was set to 100%. The raw images used to prepare Fig 3 can be found as S5 and S6 Figs.
Fig 4.
Functionality of KHV PCR-LFA prototype.
No PCR—no PCR control; -KHV—no template control; +KHV—positive control (approx. 1x105 KHV copies/μl); [C]—immunoassay control; [IAC]—internal amplification control line (black arrow); [KHV]–KHV-specific test line (red triangle). Comprehensive results are given in S2 Fig and are based on the raw image file S7 Fig.
Fig 5.
Graphical presentation of the limit of detection determined using serial dilutions of the cTL-21 plasmid standard.
Table 2.
Concentrations of the plasmid standard cTL-21 tested in five replicates using PCR-LFA.
Fig 6.
Analytical specificity of the KHV PCR-LFA.
None of the samples from viruses, bacteria, food fish, and the alga gave a KHV-specific signal. KHV—indicates the location of the KHV-specific test line. IAC—indicates the location of the internal amplification control line. General functionality of the LFA is confirmed by the appearance of the immunoassay control line—C. The raw image file is given as S8 Fig.
Table 3.
PCR-LFA results obtained for intra- and inter-assay variability.