Development and Clinical Validation of a Potential Penside Colorimetric Loop-Mediated Isothermal Amplification Assay of Porcine Circovirus Type 3

FIGURE 1. Whole detection procedure for vLAMP detection of PCV3. (A) Total DNA of PCV3 extracted from infected domestic pigs. (B) Metal bath and real-time qPCR instrument were used for qualitative and quantitative analyses separately. Neutral red chromogenic agents in reaction solutions led to color changes based on pH value as hydrogen ions released during DNA biosynthesis in positive reactions. For negative reaction, color of tube stayed same after reaction. (C) For qualitative analysis, results were directly discriminated with naked eye according to color changes, and for quantitative analysis, real-time qPCR was used to capture fluorescent signals; result was obtained from amplification curves.

FIGURE 2. Design and validation of vLAMP primers. (A) Target region of vLAMP primers is shown, and reference sequence is KY778777.1 PCV3. (B) Phylogenetic tree of PCV3 complete genome. Phylogenetic analysis was carried out to explain universality of reference sequence. Strains from different countries are marked with different colors, and reference sequence KY778777.1 is marked in large dark blue font. (C) Validation of vLAMP primers. Color change proved good performance of vLAMP primers. (a) A pink color occurred in vLAMP reaction with 106 copies/μl pUC57-PCV3, as templates indicated a positive reaction; (b) A orange color occurred in vLAMP with ddH2O, as templates indicated a negative reaction. (D) Gel electrophoresis of vLAMP products. Ladder-like bands indicated success amplification. From left to right: lane M, DNA Marker DL-2000 (Takara); lane 1, vLAMP with 106 copies/μl pUC57-PCV3 as templates; lane 2, vLAMP with ddH2O as templates.

FIGURE 3. Optimization of vLAMP reaction. Three- and two-dimensional plots of effect of interactions between fluorescence units (FUs) and variables (incubation time and reaction temperature). These plots are depiction of response surface methodology by central composite design. Minimum–maximum points of incubation time and reaction temperature that we designed were 45–75 min and 55–70°C, 13 different combinations were supplied by Design-Expert software, and FU values of different combinations obtained from real-time qPCR experiments. (A) Three-dimensional plots of effect of interactions between FU and incubation time and reaction temperature. (B) Two-dimensional plots of effect of interactions between FU and incubation time and reaction temperature.

FIGURE 4. Sensitivity and specificity of vLAMP assay. (A) Sensitivity comparison between established vLAMP and Taqman qPCR assay from plasmid standards as templates showed equal sensitivity of these two methods indicated that they revealed a similar lower limit of detection. (B) Specificity test showed good specificity of established vLAMP assay. Genomes of different viruses were added to vLAMP reaction and incubated at 60°C for 60 min. Order is as follows: (1) PCV1, (2) PCV2, (3) genotype II African swine fever virus, (4) Japanese encephalitis virus, (5) classical swine fever virus, (6) porcine reproductive and respiratory syndrome, (7) pseudorabies virus, (8) porcine parvovirus, (9) porcine epidemic diarrhea virus, (10) swine influenza virus, (11) 106 copies/μl pUC57-PCV3, and (12) ddH2O. Only sample 11 (PCV3) was positive.

FIGURE 5. Evaluation of vLAMP assay with clinical serum samples. (A) Detection results of a total of 407 pooled swine serum samples from six selected geographical regions of China using both Taqman qPCR and vLAMP were shown by scatter plot. X-axis showed CT values of TaqMan qPCR assay, and Y-axis showed TP values of vLAMP assay. (B) Swine serum sample collection areas in this study and distribution of PCV3 detected by vLAMP in China. (a) Sample collection areas in this study. (b) Thermal map based on a positive rate of PCV3.

FIGURE 6. Evaluation of direct vLAMP assay with clinical serum samples. (A) Forty-eight clinical serum samples (36 positives and 12 negatives) covering all sample types were subject to either vLAMP (red plot) or direct vLAMP assay (blue plot). Result shows most samples with a CT < 32 could be detected by both vLAMP and direct-vLAMP assay. (B) Evaluation of sensitivity and specificity of direct-vLAMP and preheated direct-vLAMP assay. To investigate if preheated serum samples could influence results, 48 serum samples were selected to evaluate specificity and sensitivity of vLAMP assay after preheated handling. Left shows that preheated have little influence on specificity of vLAMP, whereas right shows a higher sensitivity after preheated especially when samples’ CT ≥ 30. (C) Receiver operating characteristic (ROC) curves analysis to indicate accuracy of direct-vLAMP and preheated direct-vLAMP assay on samples of different CT ranges. When CT value of samples was less than 35, preheated increased sensitivity strikingly. (a) ROC curves of direct-vLAMP assay. (b) ROC curves of preheated direct-vLAMP assay.