key: cord-0880063-407bgxzx authors: Hoang, Minh; Wu, Hung‐Yi; Lien, Ying‐Xiu; Chiou, Ming‐Tang; Lin, Chao‐Nan title: A SimpleProbe(®) real‐time PCR assay for differentiating the canine parvovirus type 2 genotype date: 2018-08-31 journal: J Clin Lab Anal DOI: 10.1002/jcla.22654 sha: 2272b370fe7b1e730af1659f9e3099c451b1a21d doc_id: 880063 cord_uid: 407bgxzx BACKGROUND: Canine parvovirus type 2 (CPV‐2) causes an important canine viral disease worldwide. CPV‐2 belongs to the Protoparvovirus genus in the family Parvoviridae. An amino acid change at position 426 of the VP2 protein differentiate types of CPV‐2, designated as CPV‐2a (Asn), CPV‐2b (Asp), and CPV‐2c (Glu). In this study, we compared CPV‐2 genotyping results obtained by SimpleProbe(®) real‐time PCR and DNA sequencing analysis to identify the accuracy and sensitivity of these methods. METHODS: One hundred rectal swabs were collected from CPV‐2 naturally infected dogs from 2015 to 2017 at the Animal Disease Diagnostic Center, National Pingtung University of Science and Technology. CPV‐2 genotyping was performed by SimpleProbe(®) real‐time PCR and DNA sequencing to compare results. RESULTS: CPV‐2a (n = 23), 2b (n = 6) and 2c (n = 71) genotyping results obtained by both techniques were identical with specificity of 100% for SimpleProbe(®) assay. In the SimpleProbe(®) assay, amplifying the DNAs prepared from the clinical specimens showed three distinct melting curve peaks. CPV‐2b had the highest melting peak of 57.8°C (CI 95%: 57.7‐58.5°C) followed by CPV‐2c with a slightly lower melting peak of 52.3°C (CI 95%: 52.2‐53.2°C) and CPV‐2a with the lowest peak of 50.2°C (CI 95%: 50.1‐50.5°C). CONCLUSION: This study developed a novel method for genotyping CPV‐2 strains using the SimpleProbe(®) real‐time PCR assay. This assay is a reliable and sensitive tool for differentiating between the CPV‐2a, 2b and 2c and this technique can be used for molecular CPV‐2 epidemiology studies. diagnosis. Because a point mutation at the amino acid position 426 in the VP2 protein has been identified to be associated with the different types of CPV-2, sequencing analysis by conventional PCR can be utilized to provide details in terms of CPV typing. 4 In addition, this amino acid substitution is caused by mutating AAT (2a) to GAT (2b) at nucleotide position 1276 or to GAA (2c) at nucleotide positions 1276 and 1278 in the VP2 gene. 4 Traditionally, identifying the CPV-2 variants has been performed by hemagglutination inhibition (HI) testing using monoclonal antibodies (MAbs), 8, 18 PCR-RFLP using enzyme MboII, 4 PCR-based methods, 19 and sequence analyses. HI testing using MAbs helps to predict CPV-2 antigen specificity, which distinguishes the CPV-2 variants. Although types 2a and 2b differ in their lack of MAb B4A2 reactivity to CPV-2b, this MAb cannot recognize type CPV-2c. Thus, a MAb was developed to differentiate the new variant, 2c, from type 2b. 8 However, only samples with high HA titers (≥1:64) can be characterized using MAbs, and several samples containing high viral DNA titers that tested negative or poorly positive by HA have been calculated by real-time PCR. 20, 21 In addition, nonhemagglutinating strains have been described. 22 The PCR-RFLP assay with enzyme MboII 4 can only identify CPV-2c. Both types 2a and 2b are unrecognized by the enzyme and consequently are indistinguishable by utilizing this method; thus, this sequence analysis is often required to definitively characterize the strain, which is more expensive and time-consuming. 21 PCR-based methods have been developed to identify type 2, 2a and 2b CPVs 23 by utilizing nucleotide differences between the primers restricted to one base on their 3′ end. However, these mismatches at the 3′ end of the primers would be insufficient to prevent other CPV-2 types from amplifying. 4 In addition, among the type-specific PCR assays, CPV-2c is undetectable, as the Asp426Glu substitution is due to a change (T to A) at the third codon position at nucleotide 1278 of the VP gene, this mutant is recognized erroneously as type 2b using this PCR strategy. 4 The minor groove binder (MGB) probe real-time PCR assay was developed to rapidly and unambiguously characterize CPV-2 variants. 5 The MGB probe assays with two probe designs can recognize single nucleotide polymorphisms (SNPs) that exist between types 2b/2b (A1276G) and (T1278A), which determine the presence of amino acids, Asn, Asp and Glu, in types 2a, 2b and 2c, respectively, at residue 426 of the capsid protein. 4 Both type 2a/2b and type 2b/2c assays are highly sensitive and specific despite the type 2a-specific probe not discriminating type 2a CPVs from the original type 2. SimpleProbe contains only a single-labeled sensor probe, which can perform the SNP genotyping in human, animal, or plant samples. [24] [25] [26] [27] In this study, we compared CPV-2 genotyping results obtained by SimpleProbe ® real-time PCR and DNA sequencing analysis. One hundred rectal swabs were collected from CPV-2 naturally in- Taipei City, Taiwan) according to the manufacturer's protocol. All clinical specimens were confirmed to be infected with CPV-2 by qPCR using SYBR Green. 28 DNA sequencing was performed by PCR products, as described by Chiang et al. 7 To construct the different CPV-2 variants, a partial VP2 gene was amplified as described by Buonavoglia et al. 4 Subsequently, the control plasmids were constructed as described by Lin et al. 29 The genotypes of all of control clones were confirmed by DNA sequencing. The SimpleProbe ® (TIB MOLBIOLGmbH, Berlin, Germany) was designed based on specific features of the CPV-2b variant. The primers and probe sequences are listed in Table 1 temperature from 40°C to 75°C. The sensitivity and performance of the SimpleProbe ® assay were evaluated using 10 replications of 10-fold-diluted control plasmid from 10 2 -10 6 , from which the realtime PCR detection limit was determined. In addition, the specificity of the SimplePrboe ® assay was also assessed by testing nucleic Figure 1 ). The detection limits of the assay were evaluated using 10-fold dilution replicates of plasmids 2a, 2b and 2c, corresponding to an input equivalent to 10 2 -10 6 DNA copies/μL. The 10-fold-diluted plasmid tests showed that the SimpleProbe ® assay detected approximately 10 4 DNA copies/μL (Table 3) . Moreover, DNA extracted from canine circovirus, canine coronavirus, canine distemper virus, calicivirus, and rabies virus did not interfere with the assay ( Figure S1 ). In the SimpleProbe ® assay, amplifying the DNAs prepared from the clinical specimens showed three distinct melting curve peaks: CPV-2b had the highest melting peak of 57.8°C (CI 95%: 57.7-58.5°C) ( Figure 2B ), followed by CPV-2c with a slightly lower melting peak of 52.3°C (CI 95%: 52.2-53.2°C) ( Figure 2C ) and CPV-2a with the lowest peak of 50.2°C (CI 95%: 50.1-50.5°C) ( Figure 2A ) ( Table 2 ). All three CPV-2 sample genotypes were correctly identified from the melting curve peaks and DNA sequencing (Figure 2A-C) . All genotyping results from the SimpleProbe ® assay were identical to the DNA sequencing results (Table S1 ). The specificity of this SimpleProbe ® assay was 100% in all CPV-2 genotypes. We also evaluated ten samples that could not be evaluated by DNA sequencing due to low viral loads (cq>35). The SimpleProbe ® assay identified one samples as genotype CPV-2c, and the remaining nine samples were negative (data not shown). (Figure 3) . Both The SimpleProbe ® assay required less time for genotyping than the conventional sequencing method. Moreover, the large sample size could be tested simultaneously, and the SimpleProbe ® assay was cheaper than DNA sequencing. Overall, the SimpleProbe ® assay offers some distinct advantages for genotyping CPV-2: (a) it reduces the cost and time required for DNA sequencing; (b) data are easy to interpret; (c) only a single-labeled sensor probe is used; and (d) it offers excellent specificity. This study developed a novel method for genotyping CPV-2 strains using the SimpleProbe ® real-time PCR assay. This assay is a reliable and sensitive tool for differentiating between the CPV-2a, 2b and 2c. This technique may be useful for molecular CPV-2 epidemiology studies. We gratefully acknowledge the excellent technical assistance of Dr. Ya-Ling Lin. Evolution of canine parvovirus-A need for new vaccines? 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