A reliable RT-PCR/RFLP assay for the molecular classification of enterovirus reference and wild type strains to either of the two genetic clusters on the basis of 5′-UTR

Abstract Background The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs. Result First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. Conclusion In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

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SARS-CoV-2 variants of concern are associated with lower RT-PCR amplification cycles between January and March 2021 in France

10.1101/2021.03.19.21253971 ◽

2021 ◽

Author(s):

Benedicte Roquebert ◽

Stephanie Haim-Boukobza ◽

Sabine Trombert-Paolantoni ◽

Emmanuel Lecorche ◽

Laura Verdurme ◽

...

Keyword(s):

Pcr Amplification ◽

Wild Type ◽

Rt Pcr ◽

Cycle Threshold ◽

Type Strains

SARS-CoV-2 variants raise concern regarding the mortality caused by COVID-19 epidemics. We analyse 88,375 cycle amplification (Ct) values from variant-specific RT-PCR tests performed between January 26 and March 13, 2021. We estimate that on March 12, nearly 85% of the infections were caused by the V1 variant and that its transmission advantage over wild type strains was between 38 and 44%. We also find that tests positive for V1 and V2/V3 variants exhibit significantly lower cycle threshold (Ct) values.

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Development of an RT-PCR/RFLP assay to detectHosta virus Xin hosta (Hostaspp.)

The Journal of Horticultural Science and Biotechnology ◽

10.1080/14620316.2011.11512724 ◽

2011 ◽

Vol 86 (1) ◽

pp. 50-54 ◽

Cited By ~ 1

Author(s):

O. L. Fajolu ◽

R.-H. Wen ◽

A. S. Windham ◽

M. T. Windham ◽

M. R. Hajimorad

Keyword(s):

Rt Pcr ◽

Pcr Rflp ◽

Rflp Assay

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One-step multiplex real-time RT-PCR assay for detecting and genotyping wild-type group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in stool samples

PeerJ ◽

10.7717/peerj.1560 ◽

2016 ◽

Vol 4 ◽

pp. e1560 ◽

Cited By ~ 21

Author(s):

Rashi Gautam ◽

Slavica Mijatovic-Rustempasic ◽

Mathew D. Esona ◽

Ka Ian Tam ◽

Osbourne Quaye ◽

...

Keyword(s):

Limit Of Detection ◽

Wild Type ◽

Rt Pcr ◽

Pcr Assay ◽

Qrt Pcr ◽

Group A ◽

One Step ◽

Stool Samples ◽

Pcr Assays ◽

Type Strains

Background.Group A rotavirus (RVA) infection is the major cause of acute gastroenteritis (AGE) in young children worldwide. Introduction of two live-attenuated rotavirus vaccines, RotaTeq® and Rotarix®, has dramatically reduced RVA associated AGE and mortality in developed as well as in many developing countries. High-throughput methods are needed to genotype rotavirus wild-type strains and to identify vaccine strains in stool samples. Quantitative RT-PCR assays (qRT-PCR) offer several advantages including increased sensitivity, higher throughput, and faster turnaround time.Methods.In this study, a one-step multiplex qRT-PCR assay was developed to detect and genotype wild-type strains and vaccine (Rotarix® and RotaTeq®) rotavirus strains along with an internal processing control (Xeno or MS2 RNA). Real-time RT-PCR assays were designed for VP7 (G1, G2, G3, G4, G9, G12) and VP4 (P[4], P[6] and P[8]) genotypes. The multiplex qRT-PCR assay also included previously published NSP3 qRT-PCR for rotavirus detection and Rotarix® NSP2 and RotaTeq® VP6 qRT-PCRs for detection of Rotarix® and RotaTeq® vaccine strains respectively. The multiplex qRT-PCR assay was validated using 853 sequence confirmed stool samples and 24 lab cultured strains of different rotavirus genotypes. By using thermostablerTthpolymerase enzyme, dsRNA denaturation, reverse transcription (RT) and amplification (PCR) steps were performed in single tube by uninterrupted thermocycling profile to reduce chances of sample cross contamination and for rapid generation of results. For quantification, standard curves were generated using dsRNA transcripts derived from RVA gene segments.Results.The VP7 qRT-PCRs exhibited 98.8–100% sensitivity, 99.7–100% specificity, 85–95% efficiency and a limit of detection of 4–60 copies per singleplex reaction. The VP7 qRT-PCRs exhibited 81–92% efficiency and limit of detection of 150–600 copies in multiplex reactions. The VP4 qRT-PCRs exhibited 98.8–100% sensitivity, 100% specificity, 86–89% efficiency and a limit of detection of 12–400 copies per singleplex reactions. The VP4 qRT-PCRs exhibited 82–90% efficiency and limit of detection of 120–4000 copies in multiplex reaction.Discussion.The one-step multiplex qRT-PCR assay will facilitate high-throughput rotavirus genotype characterization for monitoring circulating rotavirus wild-type strains causing rotavirus infections, determining the frequency of Rotarix® and RotaTeq® vaccine strains and vaccine-derived reassortants associated with AGE, and help to identify novel rotavirus strains derived by reassortment between vaccine and wild-type strains.

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PCR-based Genotyping Assays to Detect Germline APC mutations Associated with Hereditary Gastrointestinal Polyposis in Jack Russell Terriers

10.21203/rs.3.rs-94557/v1 ◽

2020 ◽

Author(s):

Kyoko Yoshizaki ◽

Akihiro Hirata ◽

Hiroyuki Matsushita ◽

Naohito Nishii ◽

Mifumi Kawabe ◽

...

Keyword(s):

Mutant Allele ◽

False Negative ◽

Disease Onset ◽

Pcr Amplification ◽

Buccal Swab ◽

Wild Type ◽

Mutation Site ◽

Gastrointestinal Polyposis ◽

Pcr Rflp ◽

Rflp Assay

Abstract Background: The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell Terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline mutations in the APC gene (c.[462A>T; 463A>T]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC mutations were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs.Result: First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC mutations creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the mutation site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the mutation was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. Conclusion: In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC mutations. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

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Restriction fragment length polymorphism analysis of isolates of infectious bursal disease viruses from Turkey

Veterinární Medicína ◽

10.17221/5790-vetmed ◽

2012 ◽

Vol 48 (No. 12) ◽

pp. 359-362 ◽

Cited By ~ 1

Author(s):

G. Ozbes ◽

Ertas HB ◽

A. Muzo

Keyword(s):

Rflp Analysis ◽

Infectious Bursal Disease ◽

Polymorphism Analysis ◽

Rt Pcr ◽

Vp2 Gene ◽

Infectious Bursal Disease Viruses ◽

Pcr Rflp ◽

Bursal Disease ◽

Polymerase Chain ◽

Rflp Assay

Infectious bursal disease Virus (IBDV) specific reverse transcriptase/polymerase chain recation (RT/PCR) positive 40 broiler bursa fabricius samples obtained from a commercially reared flock were investigated for genetic diversity by PCR-RFLP assay. The assay amplifies a 743 bp fragment of the IBDV VP2 gene. The RFLP profiles of 40 of these positive samples were determined using the enzyme MboI. Most of the viruses had the same RFLP with the MboI enzyme. RFLP analysis of the isolates produced two different band profiles. The results of this study showed that little genetic heterogeneity exists among IBDV strains in a infected flock.

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Frequency of the JAK2V617F Mutation in Platelets from Essential Thrombocythemia (ET) Patients.

Blood ◽

10.1182/blood.v106.11.4972.4972 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4972-4972

Author(s):

Paula G. Heller ◽

Paola R. Lev ◽

Juan P. Salim ◽

Laura I. Kornblihtt ◽

Patricia S. Vassallu ◽

...

Keyword(s):

Colony Growth ◽

Jak2v617f Mutation ◽

Wild Type ◽

Rt Pcr ◽

Myeloid Metaplasia ◽

Pcr Rflp ◽

In The Wild ◽

Allele Specific ◽

Myelofibrosis With Myeloid Metaplasia

Abstract The JAK2V617F mutation has been recently reported in patients with polycythemia vera (PV) and a proportion of patients with ET and myelofibrosis with myeloid metaplasia. This acquired point mutation constitutively activates JAK2 tyrosine kinase and is believed to underlie growth factor hypersensitivity displayed by hematopoietic progenitors in these disorders. Identification of this genetic abnormality may be useful for diagnostic purposes although its prognostic implication remains to be determined. The prevalence of this mutation in granulocytes from ET patients ranges from 23 to 57% depending on the detection technique used. ET is characterized by predominant involvement of the megakaryocytic lineage and increased platelet production. In order to evaluate whether the frequency of the V617FJAK2 mutation in platelets from ET patients differs from that previously reported in granulocytes, we amplified a fragment of JAK2 cDNA from leukocyte-depleted platelets by RT-PCR followed by digestion with BsaXI restriction endonuclease (PCR-RFLP). The JAK2V617F mutation abolishes a BsaXI restriction site present in the wild-type sequence and generates a different band pattern. In addition, allele-specific RT-PCR (ASPCR) was performed with a primer pair common to both wild-type and mutant alleles and a third primer complementary to the mutant allele. Forty four patients with ET diagnosed according to the PVSG criteria were included, median age 39 years old (14–81), 10 were men; 21 were studied before treatment, 10 during treatment with anagrelide or hydroxyurea and 13 both before and during treatment. Twenty-two (50%) patients were found to harbour the JAK2V617F mutation by both PCR-RFLP and ASPCR, 3 were homozygous. No change in genotype was found in repeated samples from patients studied both before and during treatment. Hemoblobin levels and the frequency of thrombosis were significantly higher in patients with the mutation respect to those without (141 ±14 g/L vs. 129 ± 11 g/L, p=0.009, and 41% vs. 4.5%, p=0.004, respectively). In addition, patients carrying the mutation were significantly older (51 vs. 33 years old, p=0.002) and had lower platelet counts at diagnosis (1.070 x 109/L vs. 1.500 x 109/L, p=0.02). Interestingly, 2 patients harbouring the mutation developed PV on follow-up. In conclusion, the prevalence of the JAK2V617F mutation in platelets from ET patients did not differ from that previously reported in granulocytes. The increase in the frequency of thrombosis found in patients carrying the mutation should be confirmed in a larger study. Besides, the finding of higher hemoglobin levels in this group of patients is of interest since JAK2V617F-positive ET patients have been reported to display PV markers such as endogenous erythroid colony growth and PRV-1 overexpression, suggesting they may have larger involvement of the erythroid lineage. Further studies will help clarify whether the risk of developing PV is higher in this subset of patients.

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A Multifunction ABC Transporter (Opt) Contributes to Diversity of Peptide Uptake Specificity within the Genus Lactococcus

Journal of Bacteriology ◽

10.1128/jb.186.19.6492-6500.2004 ◽

2004 ◽

Vol 186 (19) ◽

pp. 6492-6500 ◽

Cited By ~ 25

Author(s):

Mauld Lamarque ◽

Pascale Charbonnel ◽

Dominique Aubel ◽

Jean-Christophe Piard ◽

Danièle Atlan ◽

...

Keyword(s):

Sequence Analysis ◽

Lactococcus Lactis ◽

Abc Transporter ◽

Binding Proteins ◽

Wild Type ◽

Genetic Organization ◽

Peptide Uptake ◽

Type Strains ◽

Free Strains

ABSTRACT Growth of Lactococcus lactis in milk depends on the utilization of extracellular peptides. Up to now, oligopeptide uptake was thought to be due only to the ABC transporter Opp. Nevertheless, analysis of several Opp-deficient L. lactis strains revealed the implication of a second oligopeptide ABC transporter, the so-called Opt system. Both transporters are expressed in wild-type strains such as L. lactis SK11 and Wg2, whereas the plasmid-free strains MG1363 and IL-1403 synthesize only Opp and Opt, respectively. The Opt system displays significant differences from the lactococcal Opp system, which made Opt much more closely related to the oligopeptide transporters of streptococci than to the lactococcal Opp system: (i) genetic organization, (ii) peptide uptake specificity, and (iii) presence of two oligopeptide-binding proteins, OptS and OptA. The fact that only OptA is required for nutrition calls into question the function of the second oligopeptide binding protein (Opts). Sequence analysis of oligopeptide-binding proteins from different bacteria prompted us to propose a classification of these proteins in three distinct groups, differentiated by the presence (or not) of precisely located extensions.

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