Genotyping Primers Research Articles

ProtoSpaceJAM: an open-source, customizable and web-accessible design platform for CRISPR/Cas insertional knock-in.

CRISPR/Cas-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments, currently supporting SpCas9, SpCas9-VQRand enAsCas12a Cas enzymes. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce 'recoding' mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM's design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for deep sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.

Allele-specific PCR with fluorescently labeled probes: criteria for selecting primers for genotyping

Allele-specific PCR with fluorescently labeled probes: criteria for selecting primers for genotyping

LncRNAway: a web-based sgRNA design tool for precise and effective suppression of long noncoding RNAs.

Thousands of long noncoding RNAs (lncRNAs) have been annotated via high-throughput RNA sequencing, yet only a small fraction have been functionally investigated. Genomic knockout is the mainstream strategy for studying the biological function of protein-coding genes and lncRNAs, whereas the complexity of the lncRNA locus, especially the natural antisense lncRNAs (NAT-lncRNAs), presents great challenges. Knocking out lncRNAs often results in unintended disruptions of neighboring protein-coding genes and small RNAs, leading to ambiguity in observing phenotypes and interpreting biological function. To address this issue, we launched LncRNAway, a user-friendly web tool based on the BESST (branchpoint to 3' splicing site targeting) method, to design sgRNAs for lncRNA knockout. LncRNAway not only provides specific and effective lncRNA knockout guidelines but also integrates genotyping primers and quantitative PCR primers designing, thereby streamlining experimental procedures of lncRNA function study. LncRNAway is freely available at https://www.lncrnaway.com.

An Optimized Genotyping Workflow for Identifying Highly SCRaMbLEd Synthetic Yeasts.

Synthetic Sc2.0 yeast strains contain hundreds to thousands of loxPsym recombination sites that allow restructuring of the Saccharomyces cerevisiae genome by SCRaMbLE. Thus, a highly diverse yeast population can arise from a single genotype. The selection of genetically diverse candidates with rearranged synthetic chromosomes for downstream analysis requires an efficient and straightforward workflow. Here we present loxTags, a set of qPCR primers for genotyping across loxPsym sites to detect not only deletions but also inversions and translocations after SCRaMbLE. To cope with the large number of amplicons, we generated qTagGer, a qPCR genotyping primer prediction tool. Using loxTag-based genotyping and long-read sequencing, we show that light-inducible Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination events when applied to Sc2.0 strains containing a linear or a circular version of synthetic chromosome III.

Rapid Visual Detection of Elite Erect Panicle Dense and Erect Panicle 1 Allele for Marker-Assisted Improvement in Rice (Oryza sativa L.) Using the Loop-Mediated Isothermal Amplification Method.

Molecular-assisted breeding is an effective way to improve targeted agronomic traits. dep1 (dense and erect panicle 1) is a pleiotropic gene that regulates yield, quality, disease resistance, and stress tolerance, traits that are of great value in rice (Oryza sativa L.) breeding. In this study, a colorimetric LAMP (loop-mediated isothermal amplification) assay was developed for the detection of the dep1 allele and tested for the screening and selection of the heavy-panicle hybrid rice elite restorer line SHUHUI498, modified with the allele. InDel (Insertion and Deletion) primers (DEP1_F and DEP1_R) and LAMP primers (F3, B3, FIP, and BIP) for genotyping were designed using the Primer3 Plus (version 3.3.0) and PrimerExplore (version 5) software. Our results showed that both InDel and LAMP markers could be used for accurate genotyping. After incubation at a constant temperature of 65 °C for 60 min with hydroxynaphthol blue (HNB) as a color indicator, the color of the LAMP assay containing the dep1 allele changed to sky blue. The SHUHUI498 rice line that was detected in our LAMP assay displayed phenotypes consistent with the dep1 allele such as having a more compact plant architecture, straight stems and leaves, and a significant increase in the number of effective panicles and spikelets, demonstrating the effectiveness of our method in screening for the dep1 allele in rice breeding.

Molecular Detection of Hepatits B Virus Genotypes in Tertiary Hospitals in Bayelsa State, Nigeria

In Africa, the Hepatitis B Virus (HBV) is considered endemic or even hyper-endemic. However, there is lack adequate comprehensive data regarding the genotypes of HBV and their distribution within Nigeria. This study seeks to identify the prevailing HBV genotypes among patients who sought medical care at the Federal Medical Center, Yenagoa and Niger Delta University Teaching Hospital Okolobiri, Bayelsa State, Nigeria. Between January and June 2022, 656 patients’ blood samples were collected from both hospitals for analysis, consisting of 475 females (72.4%) and 181 males (27.6%). The samples were tested for Hepatitis B surface antigen (HBsAg) and genotyping using immunochromatography and multiplex Polymerase Chain Reaction (PCR) techniques with type-specific primers. Among the 656 patients screened for HBsAg, 66 individuals (10%), comprising 36 females (5.4%) and 30 males (4.6%), tested positive using immunochromatography. These positive cases underwent molecular genotyping using specific primers for genotypes A, B, C, D, E, and F. Of these, 33 (50%) exhibited strong or active positivity. In comparison, the remaining 50% displayed passive positivity due to viral degradation, as HBV is a non-enveloped virus. The results further revealed that HBV genotypes E and B were the predominant types, with a prevalence of 82.4% and 11.8%, respectively, in the study area. Interestingly, the observation showed that co-infections involving HBV/B and HBV/E had a majority of 5.9% and were detected among two female patients aged 26 and 25. It is noteworthy that in the study area and its environs, healthcare providers commonly prescribe tenofovir, a nucleotide analog drug. Previous research by various authors has indicated that HBV genotype E is more responsive to nucleotide analogs, while HBV genotype B responds better to interferon-based therapies. In conclusion, this study highlights the prevalence of HBV genotypes B, E, and B+E coinfection in Yenagoa within the Niger Delta region of Nigeria. This knowledge underscores the importance of healthcare providers accurately diagnosing HBV genotypes before prescribing treatment regimens, particularly considering combination therapy, to optimize the care of infected patients. This approach is vital for effectively managing HBV infections and improving patient outcomes.

Epstein-Barr virus genotype-1 and Mediterranean + strain in gastric cancer biopsies of Ghanaian patients.

Gastric cancer (GC) prevalence is on the increase in Ghana, and Epstein-Barr virus (EBV) is one of the factors that have been implicated in the etiology of the cancer. It is therefore important to know the contribution of EBV genotype and strains that are associated with GC. In this study, we aimed at genotyping EBV and determining predominant strains in GC biopsies in Ghanaian patients. Genomic DNA was extracted from 55 GC biopsies (cases) and 63 normal gastric tissues (controls) were amplified by polymerase chain reaction (PCR) using specific primers for EBV detection and genotyping followed by PCR fragments sequencing. Epstein-Barr virus positivity were 67.3% and 49.2% in the GC and normal biopsies, respectively. Both cases and controls had the Mediterranean + strain of EBV. The predominant genotype of the virus in the GC cases was genotype-1 (75.7%) compared to 66.7% of genotype-2 among the control group. Infection was associated with GC in the study population (OR = 2.11, P = 0.014, 95% CI: 1.19 - 3.75), and EBV genotype-1 significantly increased the risk of GC (OR = 5.88, P < 0.0001, 95% CI: 3.18-10.88). The mean EBV load in the cases (3.507 ± 0.574) was significantly higher than in the controls (2.256 ± 0.756) (P < 0.0001). We conclude that EBV, especially Mediterranean + genotype-1, was the predominant strain in GC biopsies and GC type or progression is independent of the viral load.

NGS Technology in Monitoring the Genetic Diversity of Cytomegalovirus Strains.

The object of the study were samples of biological substrates (leukocyte mass, saliva, urine) taken from patients who underwent liver and kidney transplantation. Detection of CMV DNA was carried out by a real-time PCR using commercial diagnostic AmpliSense CMV-FL test systems (Central Research Institute for Epidemiology, Moscow, Russia). DNA extraction was performed using DNA-sorb AM and DNA-sorb V kits (Central Research Institute for Epidemiology) in accordance with manufacturer's manual. The quality of the prepared DNA library for sequencing was assessed by means of the QIAxcel Advanced System capillary gel electrophoresis system (QIAGEN, Germany). Alignment and assembly of nucleotide sequences were carried out using CLC Genomics Workbench 5.5 software (CLC bio, USA). The sequencing results were analyzed using BLAST of NCBI server. CMV DNA samples were selected for genotyping. The two variable genes, UL55(gB) and UL73(gN), were used for CMV genotype determination, which was performed using NGS technology MiSeq sequencer (Illumina, USA). Based on the exploratory studies and analysis of literature sources, primers for genotyping on the UL55(gB) and UL73(gN) genes have been selected and the optimal conditions for the PCR reaction have been defined. The results of sequencing the UL55(gB) and UL73(gN) gene fragments of CMV clinical isolates from recipients of solid organs made it possible to determine the virus genotypes, among which gB2, gN4c, and gN4b were dominant. In some cases, association of two and three CMV genotypes has been revealed. The application of the NGS technology for genotyping cytomegalovirus strains can become one of the main methods of CMV infection molecular epidemiology, as it allows for obtaining reliable results with a significant reduction in research time.

Characterization of the sex determining region of channel catfish (Ictalurus punctatus) and development of a sex-genotyping test

Channel catfish is an important species for aquaculture that exhibits a sexually dimorphic growth in favor of males. Genetic sexing and development of sex markers are crucial for the early identification of sex and of particular genotypes (YY males) for the production of all-male population in channel catfish aquaculture. In this study, we sequenced genomic DNA from pools of males and pools of females to better characterize the sex determining region (SDR) of channel catfish and to develop sex-specific markers for genetic sexing. Performing comparative analyses on male and female pooled genomic reads, we identified a large SDR (∼8.3 Mb) in the middle of channel catfish linkage group 4 (LG04). This non-recombining SDR contains a high-density of male-specific (Y chromosome) fixed single nucleotide polymorphisms (SNPs) along with ∼ 185 kb male-specific insertions or deletions. This SDR contains 95 annotated protein-encoding genes, including the recently reported putative channel catfish master sex determining (MSD) gene, breast cancer anti-estrogen resistance protein 1 (bcar1), located at one edge of the SDR. No sex-specific SNPs and/or indels were found in the coding sequence of bcar1, but one male-specific SNP was identified in its first intron. Based on this genomic information, we developed a PCR-based sex-specific genetic test. Genotyping results confirmed strong linkage between phenotypic sexes and the identified SDR in channel catfish. Our results confirm, using a Pool-Seq approach, that channel catfish is male heterogametic (XX-XY) with a large SDR on the LG04 sex chromosome. Furthermore, our genotyping primers can be used to identify XX, XY, and YY fish that will facilitate future research on sex determination and aquaculture applications in channel catfish.

ABO Genotyping from Pulp and Dentin Using DNA - A PCR Study

Background: Blood grouping has a major role in forensic science in the field of human identification. Aim of the Study: Aim of this study was to determine ABO genotyping from pulp and dentin using PCR method. Objectives: To determine PCR based blood grouping from the DNA isolated from tooth Pulp and Dentin. Materials and Meathods: Dental pulp tissue and Dentin was collected from 20 permanent teeth and DNA extraction was carried out from pulp using (modified proteinase –K method) and from dentin using phenol/chloroform(organic method). PCR procedure was carried out and samples were subjected to agarose gel electrophoresis and blood group was determined using specific PCR primers for ABO genotyping. Results: Among the 20 samples of pulp tissue, 17 samples showed accurate results in PCR method in comparison with slide agglutination method. Among the 20 samples of dentin, blood grouping from dentin was not possible as the quantity and purity of DNA from dentin samples were not optimal. Sensitivity of 85% and specificity of 50% was noticed from the samples of pulp. Conclusion: PCR was found to be an effective method in determination of blood group from teeth. Thus our study states that isolation of DNA can be done from both pulp and dentin and the blood grouping can be done from tooth pulp by PCR method. Hence PCR method can be used for identification of individuals which adds beneficiary value to forensic dentistry.

Genotyping of Human Papilloma Virus Causing Skin Wart among Iraqi Patients

The papillomaviruses are a large group of tiny, non-enveloped DNA viruses that in several differentanatomical locations can cause squamous epithelial tumor’s (warts and papilloma’s). There is a closeassociation between HPV and cervical uterine cancer . The genome is composed of 7200–8000 base pairsand is divided into three parts, early region (E) comprising of E1, E2, E4–E8 and reflecting 50 percent of theL1 and L2 genome, Genomic DNA from wart tissue samples were extracted by using gSYAN DNA mini kitextraction kit . The Real Time PCR primers for detection and genotyping of DNA Human papillomavirustype 1 and type3 and type6 based on L1 gene that designed in this study using NCBI-Genbank Sequencedatabase and primer 3 plus . The genetic study included analysis of a common DNA to HPV that was foundin 20 % of cases and DNA fragment specific to HPV type 1, 3 and 6 based on real time PCR. Type 1 HPVwas not approved to present in any sample in the study. Type 3 HPV was identified in a single case. HPV 6was identified in 8 cases, Therefore, type 6 HPV is the most common genotype, followed by type 3.

Genotyping of Giardia duodenalis in children in upper Egypt using assemblage- specific PCR technique.

Giardia duodenalis is a common gastrointestinal protozoan parasite, causing diarrheal illness in humans worldwide. Yet, the distribution of G. duodenalis genotypes among human patients and their clinical relevance remains controversial. This study aimed to detect G. duodenalis in children in Upper Egypt and identify causative genotypes and elucidate a possible correlation between genotype and clinical presentation. One hundred sixty-five children, regardless of symptoms, were tested for giardiasis. Giardia positive stool samples (40/165) were subjected to PCR amplification targeting the tpi gene with positive PCR results in only 35 cases (87.5%). Assemblage-specific amplification of genotypes (A, B, and the zoonotic E strains) revealed predominantly G. duodenalis Assemblage A (45.7%). Assemblage B and mixed A and B infections were detected in 31.4% and 22.8% of children, respectively. Assemblage E was not detected. G. duodenalis assemblage A was dominant in children who complained of diarrhea and abdominal cramps. In contrast, asymptomatic children with positive stool samples display a higher frequency of assemblage B and mixed infections. The study highlights the predominance of Giardia Assemblage A in our study locality. This study is the first for this endemic area to use the copro-PCR technique for diagnosis and genotyping of giardiasis. Study results show the value of simple species-specific primers for genotyping in communities with little access to laboratory resources. Further genetic studies are needed to clarify the association between parasite genetic diversity and patient symptomatology.

Detection and molecular characterization of rotavirus of pigs in Kerala, India.

Group A rotaviruses (GAR) are an important cause of diarrhoea in infants and newborn animals especially pigs. In this paper, we report the detection, G and P typing and phylogenetic analysis of GAR of pigs in Kerala. A total of 100 fecal samples from diarrhoeic piglets were collected from organized farms in Wayanad, Ernakulam, Thrissur, and Palakkad districts of Kerala. The samples were tested for the presence of GAR employing reverse transcriptase polymerase chain reaction (RT-PCR) targeting VP6 gene. Positive samples were tested by G and P genotyping primers and representative amplicons were sequenced. Of the 100 samples, 12 were positive for GAR. The G and P types detected were G2, G4, G5, G6, G9, P[6] and P[19]. An untypable P type (P21-5 like) was also detected. In some of the samples more than one G type was detected. The nucleotide sequences of G2, G4 and G5 types were similar to those seen in pigs and that of G6 was similar to bovine sequences. G9, P[6] and P[19] sequences showed similarity to human rotavirus sequences. The findings of this study provide the first information on the G and P genotypes of GAR of pigs in Kerala.

Comparative Genomic and Transcriptomic Analyses of Mycobacterium kansasii Subtypes Provide New Insights Into Their Pathogenicity and Taxonomy.

Mycobacterium kansasii is an important opportunistic pathogen of humans and has a close phylogenetic relationship with Mycobacterium tuberculosis. Seven subtypes (I–VII) have been identified using molecular biology approaches, of which subtype I is the most frequent causative agent of human disease. To investigate the genotypes and pathogenic components of M. kansasii, we sequenced and compared the complete base-perfect genomes of different M. kansasii subtypes. Our findings support the proposition that M. kansasii “subtypes” I-VI, whose assemblies are currently available, should be considered as different species. Furthermore, we identified the exclusive presence of the espACD operon in M. kansasii subtype I, and we confirmed its role in the pathogenicity of M. kansasii in a cell infection model. The espACD operon is exclusively present in mycobacterial species that induce phagosomal rupture in host phagocytes and is known to be a major determinant of ESX1-mediated virulence in pathogenic mycobacteria. Comparative transcriptome analysis of the M. kansasii I-V strains identified genes potentially associated with virulence. Using a comparative genomics approach, we designed primers for PCR genotyping of M. kansasii subtypes I-V and tested their efficacy using clinically relevant strains of M. kansasii.

Genetic analysis of Ghanaian G1P[8] and G9P[8] rotavirus A strains reveals the impact of P[8] VP4 gene polymorphism on P-genotyping.

The World Health Organisation rotavirus surveillance networks have documented and shown eclectic geographic and temporal diversity in circulating G- and P- genotypes identified in children <5 years of age. To effectively monitor vaccine performance and effectiveness, robust molecular and phylogenetic techniques are essential to detect novel strain variants that might emerge due to vaccine pressure. This study inferred the phylogenetic history of the VP7 and VP4 genes of previously non-typeable strains and provided insight into the diversity of P[8] VP4 sequences which impacted the outcome of our routine VP4 genotyping method. Near-full-length VP7 gene and the VP8* fragment of the VP4 gene were obtained by Sanger sequencing and genotypes were determined using RotaC v2.0 web-based genotyping tool. The genotypes of the 57 rotavirus-positive samples with sufficient stool was determined. Forty-eight of the 57 (84.2%) had the P[8] specificity, of which 43 (89.6%) were characterized as P[8]a subtype and 5 (10.4%) as the rare OP354-like subtype. The VP7 gene of 27 samples were successfully sequenced and their G-genotypes confirmed as G1 (18/27) and G9 (9/27). Phylogenetic analysis of the P[8]a sequences placed them in subcluster IIIc within lineage III together with contemporary G1P[8], G3P[8], G8P[8], and G9P[8] strains detected globally from 2006–2016. The G1 VP7 sequences of the study strains formed a monophyletic cluster with African G1P[8] strains, previously detected in Ghana and Mali during the RotaTeq vaccine trial as well as Togo. The G9 VP7 sequences of the study strains formed a monophyletic cluster with contemporary African G9 sequences from neighbouring Burkina Faso within the major sub-cluster of lineage III. Mutations identified in the primer binding region of the VP8* sequence of the Ghanaian P[8]a strains may have resulted in the genotyping failure since the newly designed primer successfully genotyped the previously non-typeable P[8] strains. In summary, the G1, G9, and P[8]a sequences were highly similar to contemporary African strains at the lineage level. The study also resolved the methodological challenges of the standard genotyping techniques and highlighted the need for regular evaluation of the multiplex PCR-typing method especially in the post-vaccination era. The study further highlights the need for regions to start using sequencing data from local rotavirus strains to design and update genotyping primers.

Correction: GGCX and VKORC1 inhibit osteocalcin endocrine functions.

Vol. 208, No. 6, March 16, 2015. [10.1083/jcb.201409111][1]. The authors noticed a mistake in the labeling of the genotyping primers in Fig. S1 A and Table S1. In Fig. S1 A, the primers P1 and P2 are actually flanking the 5′loxP site region. In addition, the P3 primer should appear downstream of

Polymorphism rs7895833 in the SIRT1 gene and its association with dyslipidaemia in the elderly

ObjectivesSirtuin 1 is a human protein involved in gene silencing and in inducing the deacetylation of proteins involved in the metabolic and adaptive response mechanisms. Polymorphisms in the SIRT1 gene have been studied with respect to aging. This study aims to determine the allelic and genotypic frequencies of the rs7895833 A/G polymorphism in the SIRT1 gene, and to identify the association between this polymorphism and the co-morbidities prevalent in the elderly population. Material and methodsA total of 216 patients were evaluated in an outpatient clinic in Central Brazil. The individuals underwent validated tests for cognitive impairment and falls risk, serum biochemistry analysis, as well as polymer chain reaction (PCR) with confronting two-pair primers for polymorphism genotyping. Resultsrs7895833 polymorphism in SIRT1 gene was observed in these patients as follows: AA (56/216), AG (138/216), and GG (22/216). The frequency of allele A was 0.58, and that of allele G was 0.42. In the multivariate analysis of the exploratory variables, glucose, high density lipoprotein (HDL) cholesterol, systemic arterial hypertension, dyslipidaemia, and depression, which were associated in the univariate analysis with the polymorphism rs7895833, only dyslipidaemia showed a statistically significant difference in a greater number of individuals with this polymorphism. ConclusionThe variant allele G of the SIRT1 gene polymorphism was found in 42% of these Brazilian geriatric patients, and was associated with dyslipidaemia. Further studies should be performed to confirm this result and to elucidate the role of SIRT1 in lipid metabolism.

Alternative PCR primers for genotyping of Brazilian WSSV isolates.

White spot syndrome virus (WSSV) is one of the major challenges faced by global shrimp farming in recent decades. The characterization of WSSV genetic variability has been used to determine viral dispersion and is a promising method to determine the association between genotype and virulence. The major variable regions that have been used as markers to differentiate the WSSV genomes include the VNTR loci inside ORF94, ORF75, ORF125, and insertions/deletions interspersing ORF14/15 and ORF23/24. The primers used to amplify these regions were described at least 10 years ago, but some of them do not work efficiently to identify new WSSV variants. The objective of this work was to develop improved PCR primers for WSSV genotyping based on sequence alignments that include new sequences described in recent years. We validated these new primers in a pilot study to verify the genetic variability of the WSSV in Rio Grande do Norte state (northeast Brazil), and efficiency was compared to that of other previously described primers. We confirmed that the primers we developed were more efficient for genotype Brazilian WSSV isolates, enabling us to genotype a larger number of samples. In addition, our results also introduce new data about the genetic characterization of the WSSV isolates that occur in the northeastern region of Brazil.

Identification of Amino Acid Substitutions Within the VP7 Genes of G2 Rotavirus Strains in Ghana.

We used the dideoxynucleotide chain termination method to determine the strains of nine non-typeable rotavirus enzyme immunoassay-positive samples, which were identified as G2. We detected nucleotide changes in the primer-binding region and amino acid substitutions within the VP7 protein of the G2 rotavirus strains. Genotyping primers need to be updated regularly.

Genetic diversity of human sapovirus across the Americas

BackgroundSapoviruses are responsible for sporadic and epidemic acute gastroenteritis worldwide. Sapovirus typing protocols have a success rate as low as 43% and relatively few complete sapovirus genome sequences are available to improve current typing protocols. Objective/study designTo increase the number of complete sapovirus genomes to better understand the molecular epidemiology of human sapovirus and to improve the success rate of current sapovirus typing methods, we used deep metagenomics shotgun sequencing to obtain the complete genomes of 68 sapovirus samples from four different countries across the Americas (Guatemala, Nicaragua, Peru and the US). ResultsVP1 genotyping showed that all sapovirus sequences could be grouped in the four established genogroups (GI (n = 13), GII (n = 30), GIV (n = 23), GV (n = 2)) that infect humans. They include the near-complete genome of a GI.6 virus and a recently reported novel GII.8 virus. Sequences of the complete RNA-dependent RNA polymerase gene could be grouped into three major genetic clusters or polymerase (P) types (GI.P, GII.P and GV.P) with all GIV viruses harboring a GII polymerase. One (GII.P-GII.4) of the new 68 sequences was a recombinant virus with the hotspot between the NS7 and VP1 regions. ConclusionsAnalyses of this expanded database of near-complete sapovirus sequences showed several mismatches in the genotyping primers, suggesting opportunities to revisit and update current sapovirus typing methods.