key: cord-313004-gdnaiodj
authors: Wang, Yong; Cui, Yongqiu; Li, Yeqiu; Jiang, Shudong; Liu, Hua; Wang, Jing; Li, Yongdong
title: Simultaneous detection of duck circovirus and novel goose parvovirus via SYBR green I-based duplex real-time polymerase chain reaction analysis
date: 2020-08-14
journal: Molecular and Cellular Probes
DOI: 10.1016/j.mcp.2020.101648
sha: 
doc_id: 313004
cord_uid: gdnaiodj

Abstract Beak atrophy and dwarfism syndrome (BADS) is commonly caused by co-infection with duck circovirus (DuCV) and novel goose parvovirus (NGPV). Therefore, concurrent detection of both viruses is important for monitoring and limiting BADS, although such a diagnostic test has not been reported. In this study, we developed a duplex, SYBR Green I-based real-time polymerase chain reaction (PCR) assay to enable the simultaneous detection of DuCV and NGPV. The assay readily distinguished between the two viruses, based on their different melting temperatures (Tm), where the Tm for DuCV was 80 °C and that for NGPV was 84.5 °C. Other non-target duck viruses that were tested did not show melting peaks. The detection limit of the duplex assay was 101 copies/μL for both viruses. This method exhibited high repeatability and reproducibility, and both the inter-assay and intra-assay variation coefficients were <1.6%. Thirty-one fecal samples were collected for clinical testing using real-time PCR analysis, and the results were confirmed using sequencing. The rate of co-infection was 6.5%, which was consistent with the sequencing results. This duplex real-time PCR assay offers advantages over other tests, such as rapid, sensitive, specific, and reliable detection of both viruses in a single sample, which enables the quantitative detection of DuCV and NGPV in clinical samples. Using this test may be instrumental in reducing the incidence of BADS and the associated economic losses in the duck and goose industries.

circovirus (DuCV) and novel goose parvovirus (NGPV). Therefore, concurrent detection of both 29

viruses is important for monitoring and limiting BADS, although such a diagnostic test has not 30 been reported. In this study, we developed a duplex, SYBR Green I-based real-time polymerase 31 chain reaction (PCR) assay to enable the simultaneous detection of DuCV and NGPV. The assay 32 readily distinguished between the two viruses, based on their different melting temperatures (Tm), 33

where the Tm for DuCV was 80°C and that for NGPV was 84.5°C. Other non-target duck viruses 34 that were tested did not show melting peaks. The detection limit of the duplex assay was 10 1 35 copies/µL for both viruses. This method exhibited high repeatability and reproducibility, and both 36 the inter-assay and intra-assay variation coefficients were <1.6%. Thirty-one fecal samples were 37 collected for clinical testing using real-time PCR analysis, and the results were confirmed using 38

sequencing. The rate of co-infection was 6.5%, which was consistent with the sequencing results. 39

Primers for amplifying DuCV were synthesized based on a conserved region in the rep gene 116

[7], where the sequence of the forward primer was 5′-GTATCACTGACCGTTACC-3′ and that of 117 the reverse primer was 5′-GCTCTTTGTTGCTAGTTATG-3′. The primer pair was used to amplify 118 a partial region of the DuCV rep gene, with a length of 93 base pairs (bp). The primers used to 119 amplify the NGPV were synthesized based on the VP1 gene [27] , where the sequence of the 120 forward primer was 5′-AATTTCAATGAGGTAGACA-3′ and that of the reverse primer was 121 5′-ATTACTCCAGATGTTCCA-3′. The primer pair was used to amplify a partial region of the 122 NGPV VP1 gene, with a length of 171 bp. The primers were synthesized by Sangon Biotech. The duplex DuCV and NGPV SYBR Green I-based quantitative real-time PCR assay was 127 optimized by modifying the PCR thermocycling conditions, as well as the primer and SYBR (Bio-Rad, Hercules, CA, USA) using the following thermocycling program: 95°C for 15 min, 136 followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. Fluorescence signals were automatically 137 collected at the end of each cycle. 138

The singular real-time PCR assays for DuCV and NGPV were combined into one reaction 139 system containing SuperReal PreMix Plus, primers for both the viruses, and templates. The duplex 140 reaction system was then optimized using different volumes of primers, and the optimal volumes 141 of the templates were determined. Melting-curve analysis was performed to determine the 142 melting-temperature (Tm) values of the DuCV-and NGPV-specific amplicons, in order to 143 distinguish between each product. Standard, unknown, and negative-control samples were 144 analyzed in duplicate in neighboring wells, and the results are presented as average Tm values 145 from duplicate wells. 146 147 2.7. Sensitivity, specificity, and repeatability of the duplex real-time PCR assay 148

To determine the limit of detection of the duplex real-time assay, we performed real-time 149 PCR for both viruses separately, using 10-fold serial dilutions of standard plasmid templates with 150 concentrations ranging from 1 × 10 8 to 1 × 10 1 copies/µL. For the duplex real-time PCR assays, 151 we detected each virus separately or both viruses together. The lowest concentration of each 152 plasmid DNA template for which the detection rate was ≥95% was defined as the limit of 153 detection in each case. Several concentrations of the standard DuCV and NGPV plasmids (10 7 , 10 5 , and 10 3 160 copies/µL) were tested to evaluate the reproducibility of the duplex real-time PCR assay. The showed high similarities with those for references strains. Then, the recombinant plasmids were 178 using the standard DuCV and NGPV plasmids at dilutions ranging from 10 10 to 10 3 copies/μL. 180

The standard curve equation for the DuCV plasmid was y = -3.343x + 44.113, with an R 2 value of 181 0.998 (Fig. 1b) . The standard curve equation for the NGPV plasmid was y = -3.538x + 46.736, 182

with an R 2 value of 0.999 (Fig. 1c) . 183 184

Melting-curve analysis was performed with the duplex real-time PCR assay to distinguish 186 between the amplification of DuCV and NGPV. Distinct melting-curve peaks were observed for 187 DuCV (Tm: 80.5 ± 0.5°C) and NGPV (Tm: 85 ± 0.5°C), as shown in Fig. 2a and Fig. 2b , 3.3. Sensitivity, specificity, and reproducibility of the duplex real-time PCR assay ranging from 1 × 10 8 to 1 × 10 1 copies/µL, we found that the real-time assay could detect positive 201 samples at the lowest concentration (1 × 10 1 copies/µL) ( Fig. 4a and 4b) . 202

The duplex real-time PCR assay was tested with samples positive for DuCV and NGPV, or 203 other duck pathogens (NDV, DPV, MDPV, and DuAstV). As shown in Fig. 5 , the target viruses 204

were detected and other pathogens were negative, indicating that the duplex real-time PCR assay 205 showed good specificity. 206

To assess the assay reproducibility, three parallel experiments were performed with the 207 DuCV and NGPV standard plasmids at each dilution. The intra-and inter-assay CV values for 208

DuCV ranged from 0.42% to 0.98% and 0.73% to 1.52%, respectively, and the intra-and 209

inter-assay CV values for NGPV ranged from 0.34% to 0.86% and 0.69% to 1.57%, respectively 210 (Table 1) . Thus, the duplex real-time PCR assay was reliable and showed high reproducibility for 211 detecting DuCV and NGPV in the same tube. There results were in accordance with the sequencing results. The results of our duplex real-time 218 PCR assay were in 100% agreement with singular real-time PCR. Thus, the duplex real-time PCR 219 assay described in this study can be applied for the differential diagnosis of DuCV and NGPV in 220 duck farms with high specificity, sensitivity, and reproducibility.

Cross-species virus transmission from wildlife reservoirs poses remarkable health risks to 224 humans and animals, as exemplified by the current coronavirus pandemic. After several 225 cross-species spreads, the coronavirus eventually infected humans, resulting in serious effects in 226 terms of morbidity and mortality [29] [30] [31] [32] . Many other viruses can also cross species barriers, 227 including parvoviruses and circoviruses. Parvoviruses have been found in geese, ducks, and 228 canines, and circoviruses have been found in ducks, swine and canines [19, [33] [34] [35] [36] [37] . 229

DuCV [13]. However, NGPV showed a low detection rate in samples from ducks with typical 231 BADS symptoms in a subsequent study [22] . As an immunosuppressive virus, DuCV can reduce 232 the body's resistance to invading viruses; thus, when ducks are simultaneously infected with both 233 viruses, the toxic effects of NGPV can be greatly enhanced, which increases the risk of ducks 234 developing BADS [2, 11]. To further study whether co-infection with NGPV and DuCV increases 235 the risk of ducks developing BADS, there is an urgent need for an assay that enables the 236 simultaneous detection of both viruses [2, 11, 16, 21] . SYBR Green I-based assays are cheaper 237 than TaqMan assays and offer similar rapidity, specificity, sensitivity, and reproducibility when 238 combined with melting-curve analysis. These factors made SYBR Green I-based detection a good 239 choice for our duplex real-time PCR assay. Moreover, SYBR Green I-based real-time PCR had 240 been widely used as a diagnostic method because of its apparent advantages over conventional 241 PCR in terms of its simplicity, efficient detection, and sensitivity, which enable high-throughput 242 screening even in the presence of fewer copies of a viral genome [38] . 243

Duplex SYBR Green I-based real-time PCR has been used in numerous applications. In 244 general, duplex real-time PCR offers many benefits as a detection method. Accurate detection at a 245 low concentration enabled the diagnosis and prevention of porcine diarrhea at an early stage. 246

Some virulent strains may cause severe symptoms at a low titer and, thus, a sensitive detection 247 method is indispensable in such situations. SYBR Green I-based assays are more rapid, can be 248 implemented more simply, generally enable detection in a single step, and produce results that are 249 consistent with those of other methods, which reflects the greater stability of SYBR Green I-based 250 methods [39, 40] . The model exemplified in this study can be extended to the differential diagnosis 251 of other mixed viral infections, which highlights its general significance and applicability to 252 multiple areas of veterinary medicine [41] . 253

In this study, a duplex SYBR Green I-based real-time PCR assay was successfully developed 254 to amplify the rep gene of DuCV and the VP1 gene of NGPV. This assay easily distinguished both 255 two viruses based on their different Tm values, which were 85 ± 0.5°C for NGPV and 80.5 ± 256 0.5°C for DuCV. Furthermore, the detection limit of the assay was 10 1 copies/µL in both cases, 257 which renders it more sensitive than the conventional PCR technique. Thirty-one fecal samples 258 were collected to study the prevalence of both viruses, and the co-infection rate was found to be 259 which were serially diluted 10-fold over concentrations ranging from 1 × 10 10 to 1 × 10 3 copies/μL. 406

The coefficient of determination (R 2 ) and the line equation of the regression curve (y) were which were serially diluted 10-fold over concentrations ranging from 1 × 10 10 to 1 × 10 3 copies/μL. 

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