key: cord-029775-mntcor5d
authors: Oka, Tomoichiro; Yamamoto, Seiji P.; Iritani, Nobuhiro; Sato, Shigenori; Tatsumi, Chika; Mita, Tetsuo; Yahiro, Shunsuke; Shibata, Shinichiro; Wu, Fang-Tzy; Takagi, Hirotaka
title: Polymerase chain reaction primer sets for the detection of genetically diverse human sapoviruses
date: 2020-07-27
journal: Arch Virol
DOI: 10.1007/s00705-020-04746-9
sha: 
doc_id: 29775
cord_uid: mntcor5d

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.

For the initial reactivity test, double-stranded DNA fragments (gBlocks® Gene Fragments) including the sequence targeted by the PCR primers (approximately 1700 bp each) of the human sapovirus genotypes GI.1 (GenBank accession number X86560), GI.2 (AB614356), GI.3 (AB623037), GI.4 (AJ606693), GI.5 (DQ366345), GI.6 (AJ606694), GI.7 (AB522390), GII.1 (AJ249939), GII.2 (AY237420), GII.3 (AB630068), GII.4 (AB522397), GII.5 (LC190463), GII.6 (AY646855), GII.7 (AB630067), GII.8 (KX894315), GIV.1 (DQ058829), GV.1 (DQ366344), and GV.2 (AB775659) were synthesized (Integrated DNA Technologies, Coralville, IA), and 1.0 × 10 4 copies of these fragments were used.

To confirm the reactivity against clinical specimens, random-hexamer-primed cDNA synthesized from stool suspensions positive for sapoviruses (GI.1-7, GII.1-5 and GII.7 and GII.8, GIV.1, and GV.1-2) as well as stool suspensions positive for noroviruses (GI, GII), astrovirus types I and IV, and rotavirus groups A and C and DNA purified from stool suspensions positive for adenovirus type 40/41 that had been used in a previous study and stored at −30°C [11] , were tested.

PCR assays were performed using 20 µL of a reaction mixture containing 1 µL of synthetic double-stranded DNA or 2 µL of cDNA or DNA from clinical specimens, 10 µL of KAPA2G Fast HotStart ReadyMix with dye (KAPA Biosystems, Wilmington, MA), and 10 pmol of each primer. PCR amplification was performed using a GeneAmp PCR System 2700 (Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation at 95°C for 5 min, followed by 50 cycles of 95°C for 20 s, 48°C or 53°C for 20 s, and 72°C for 5 s, and a final extension at 72°C for 1 min. The PCR reaction time was approximately 1.5 h.

Seven µL of each PCR reaction mixture was analyzed by electrophoresis in a 2% agarose gel containing 40 mM Tris-acetate, 1 mM EDTA and 0.5 µg of ethidium bromide per mL, and the gel image was captured using a FAS V Gel Documentation System (Nippon Genetics, Tokyo, Japan).

Eleven PCR assays with different primer combinations were performed using a synthetic DNA panel. As shown in Fig. 1A (left panels), seven of the PCR assays, including those using the primers SV-F11 and SV-R1; SV-F21 and SV-R2; SLV5317 and SLV5749; SaV124F, SaV-1F, SaV-5F, SV-R13, and SV-R14; SaV1245Rfwd and SV-R2; SV-F13, SV-F14, SV-R13, and SV-R14; and SV-F22 and SV-R2 [12] [13] [14] [15] , either did not amplify all of the human sapovirus genotypes tested or generated PCR products with unusual sizes (i.e., GI.5 sapovirus with the SV-F11 and SV-R1 primer set) under the test conditions.

In contrast, four assays including the primer combinations i) SV-F13, SV-F14, SV-G1-R, SV-G2-R, SV-G4-R, and SV-G5-R [13] , ii) M13F-SaV1245Rfwd, M13R-SV-G1-R, M13R-SV-G2-R, M13R-SV-G4-R, and M13R-SV-G5-R, iii) M13F-HuSaV-F1, M13F-HuSaV-F2, M13F-HuSaV-F3, and M13R-HuSaV-5498R, and iv) M13F-HuSaV-5159F and M13R-HuSaV-5498R amplified all the tested human sapovirus genotypes (Fig. 1A , right panels).

As shown in Fig. 1B , these four PCR assays gave similar results when cDNA prepared from sapovirus-positive clinical specimens ranging from approximately 10 7 to 10 2 copies/reaction (Ct value range: 18-37/reaction determined by real-time RT-PCR) representing the 17 human sapovirus genotypes (GI.1-7, GII.1-5 and GII.7 and GII.8, GIV.1, and GV.1-2) was used [11] . Samples with Ct values >30 (approximately 10 2 to 10 3 copies/reaction) were only slightly detectable or difficult to detect, depending on the assay used.

No cross-reactivity was observed with specimens that were positive for norovirus GI and GII, human astrovirus types I and IV, rotavirus A and C, or adenovirus type 40/41 in the four PCR assays (data not shown).

The locations of the primers used for the four PCR assays are shown in Fig. 2 .

The PCR assay including six primers (SV-F13, SV-F14, SV-G1-R, SV-G2-R, SV-G4-R, and SV-G5-R) reported by Okada et al. was originally designed to generate PCR products of different lengths, depending on the genogroup: GI (500 bp), GII (430 bp), GIV (360 bp), and GV (290 bp) [13] (Fig. 1A , right panel, Fig. 1B, Fig. 2 ). This assay has been used in several studies to detect sapoviruses in fecal specimens [16] [17] [18] . In the current study, we confirmed that these primer combinations detected all 18 human sapovirus genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) using cDNA and/or synthetic DNA ( Table 1) .

The PCR assay including five primers (M13F-SaV1245Rfwd, M13R-SV-G1-R, M13R-SV-G2-R, M13R-SV-G4-R, and M13R-SV-G5-R) also generated PCR products of different sizes depending on the genogroup: GI (450 bp), GII (380 bp), GIV (310 bp), and GV (240 bp) (Fig. 1A , right panel, Fig. 1B, and Fig. 2) .

The assay including four primers (M13F-HuSaV-F1, M13F-HuSaV-F2, M13F-HuSaV-F3, and M13R-HuSaV-5498R) amplified similar-sized PCR products (approximately 460 bp) independently of the genogroup and genotype (Fig. 1A, right panel, and Fig. 1B) . The target regions of HuSaV-F1, -F2, and -F3 recently designed as forward primers in a broadly reactive real-time PCR for human sapoviruses [11] are similar to those of SV-F13 and SV-F14 (Fig. 2) , and the sapovirus-specific sequence of M13R-HuSaV5498R (5′-CCCCANCCNGCVHACAT-3′) ( [11] are shown in parentheses. The primer combinations and PCR product size(s) are indicated at the top and right side, respectively, of each gel image. The annealing temperature is indicated in parentheses after the primer names. M, DNA size marker 100 bp DNA ladder (New England Biolabs); NC, negative control without template CAC AT-3′) and SVR-DS6 (5′-CCCCAMCCMGCMMA-CAT-3′) primers reported by Sano et al. [19] . The assay including two primers (M13F-HuSaV-5159F and M13R-HuSaV-5498R) generated similar-sized PCR products (approximately 380 bp) for all of the sapovirus genotypes tested (Fig. 1A, right panel, and Fig. 1B) . The sapovirus-specific sequence of M13F-HuSaV-5159F was identical to that of 1245Rfwd [15] except for 1 nt (Fig. 2 and Table 1 ), and they shared the same complementary sequence in the reverse primers (HuSaV-R, and SaV1245R, respectively) used in real-time PCR assays for detection of human sapoviruses [5, 11] .

In primer-dependent PCR assays, the selection of broadly reactive primer sets is important for detecting genetically diverse human sapoviruses. In this study, seven out of 11 primer combinations, including widely used primer sets, did not detect all of the sapovirus genotypes tested (Fig. 1A and  B) . This would cause a bias towards the detected strains or cause several genotypes to be missed when used for surveillance. We recently reported an improved broad-range real-time RT-PCR assay that detected all 18 of the currently identified human sapovirus genotypes [11] . This assay can be used as a screening tool, but the amplicon size (approximately 100 bp) is too short for further genetic characterization by sequence analysis. Selection and combination of the human-sapovirus-targeting, broad-range RT-PCR assays described in this report can be used to confirm real-time RT-PCR results and an alternative screening tool to identify genetically diverse human sapoviruses in samples. An assay that distinguishes genogroups based on PCR product size will be useful in cases in which a sample includes sapoviruses of different genogroups. New sapovirus strains assigned as GII.NA1 were reported recently [20] . Based on their nucleotide sequences, the primers used in the four broadly reactive PCR assays would probably also recognize this new candidate genotype ( Fig. 2 and Table 1 ), although experimental confirmation is required.

Funding This work was supported by a grant from the Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development (AMED) (JP19fk0108049 and JP20fk0108139). The funding body had no role in study design, analysis and interpretation of data, or the decision to submit the article for publication.

Conflict of interest There are no conflicts of interest among the authors.

The ethics committee of the National Institute of Infectious Diseases deemed that human subject regulations did not apply to this study because it only used extracted and stored virus nucleic acids (Receipt number 1113).

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