Diagnostic validation of a rapid and field-applicable PCR-lateral flow test system for point-of-care detection of cyprinid herpesvirus 3 (CyHV-3)

Abstract

Koi herpesvirus disease (KHVD) is a highly infectious disease leading to outbreaks and mass mortality in captive and free-ranging common carp and koi carp. Outbreaks may result in high morbidity and mortality which can have a severe economic impact along the supply chain. Currently, control and prevention of KHVD relies on avoiding exposure to the virus based on efficient hygiene and biosecurity measures. An early diagnosis of the disease is crucial to prevent its spread and to minimize economic losses. Therefore, an easy-to-handle, sensitive, specific and reliable test prototype for a point-of-care detection of KHV was developed and evaluated in this study. We used a multiplex-endpoint-PCR followed by a specific probe hybridization step. PCR-products/hybridization-products were visualized with a simple and universal lateral flow immunoassay (PCR-LFA). Fifty-four gill tissue samples (KHV-positive n = 33, KHV-negative n = 21) and 46 kidney samples (KHV-positive n = 24, KHV-negative n = 22) were used to determine diagnostic sensitivity and specificity of the PCR-LFA. In addition, the usability of PCR-LFA to detect CyHV-3-DNA in gill swabs taken from 20 perished common carp during a KHVD-outbreak in a commercial carp stock was examined. This assay gave test results within approximately 60 min. It revealed a detection limit of 9 KHV gene copies/μl (95% probability), a diagnostic specificity of 100%, and diagnostic sensitivity of 94.81% if samples were tested in a single test run only. PCR inhibition was noticed when examining gill swab samples without preceding extraction of DNA or sample dilution. Test sensitivity coud be enhanced by examining samples in five replicates. Overall, our PCR-LFA proved to be a specific, easy-to-use and time-saving point-of-care-compatible test for the detection of KHV-DNA. Regarding gill swab samples, further test series using a higher number of clinical samples should be analyzed to confirm the number of replicates and the sample processing necessary to reveal a 100% diagnostic sensitivity.

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. Cross primer-dimer analysis.

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 [–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.

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.