A highly sensitive semi-nested real-time PCR utilizing oligospermine-conjugated degenerate primers for the detection of diverse strains of small ruminant lentiviruses

Chassalevris, Taxiarchis; Chaintoutis, Serafeim C.; Apostolidi, Evangelia; Giadinis, Nektarios D.; Vlemmas, I.; Brellou, Georgia; Dovas, Chrysostomos Ι.

doi:10.1016/j.mcp.2020.101528

Cited by 18 publications

(22 citation statements)

References 65 publications

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“…Molecular diagnosis by PCR may add diagnostic value to serodiagnosis since seronegative animals may show PCR positive results due to low antibody production [12,13]. New molecular methods are being described focused on the design of universal primers, thereby increasing sensitivity to enable the identification and removal of animals with low viral load in vivo [14][15][16][17].…”

Section: Introductionmentioning

confidence: 99%

Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping

Ramírez

Echeverría

Benito³

et al. 2021

Pathogens

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Small ruminant lentiviruses (SRLV) cause an incurable multiorganic disease widely spread in sheep and goats that disturbs animal welfare and production. In the absence of a vaccine, control measures have been traditionally based on early diagnosis and breeding with virus-inactivated colostrum with segregation of seropositive animals. However, antigenic heterogeneity, poor antibody production due to low viral load, and single strain design of most available ELISA, pose a threat to SRLV diagnosis. Genome-wide association studies have described TMEM154 E35K polymorphism as a good genetic marker for selection of resistant animals in some American and European breeds. In this study, a multitargeted serological and virological screening of more than 500 animals from four different breeds (latxa, raza Navarra, assaf, and churra) attending to SRLV infection status was performed. Then, animals were genotyped to characterize TMEM154 E35K polymorphism. ELISA procedures, individually considered, only identified a proportion of the seropositive animals, and PCR detected a fraction of seronegative animals, globally offering different animal classifications according to SRLV infection status. TMEM154 allele frequency differed substantially among breeds and a positive association between seroprevalence and TMEM154 genotype was found only in one breed. Selection based on TMEM154 may be suitable for specific ovine breeds or SRLV strains, however generalization to the whole SRLV genetic spectrum, ovine breeds, or epidemiological situation may need further validation.

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Section: Introductionmentioning

confidence: 99%

Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping

Ramírez

Echeverría

Benito³

et al. 2021

Pathogens

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“…To achieve sufficient specificity, the primers have to be designed for conserved regions of the viral genome, avoiding the env gene which is less conserved among genotypes [ 18 , 90 ]. On the other hand, the problem of virus genetic variability can be mitigated by the use of degenerate primers expanding the detection range and improving the sensitivity of the method [ 80 , 84 , 91 ]. In infected animals, false negative results of PCR could be linked to co-existence of multiple SRLVs mutants in an infected population.…”

Section: Diagnosis Of Small Ruminant Lentiviral Infectionmentioning

confidence: 99%

Serological, Molecular and Culture-Based Diagnosis of Lentiviral Infections in Small Ruminants

Kalogianni

Stavropoulos

Chaintoutis

et al. 2021

Viruses

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Small ruminant lentiviruses (SRLVs) infections lead to chronic diseases and remarkable economic losses undermining health and welfare of animals and the sustainability of farms. Early and definite diagnosis of SRLVs infections is the cornerstone for any control and eradication efforts; however, a “gold standard” test and/or diagnostic protocols with extensive applicability have yet to be developed. The main challenges preventing the development of a universally accepted diagnostic tool with sufficient sensitivity, specificity, and accuracy to be integrated in SRLVs control programs are the genetic variability of SRLVs associated with mutations, recombination, and cross-species transmission and the peculiarities of small ruminants’ humoral immune response regarding late seroconversion, as well as intermittent and epitope-specific antibody production. The objectives of this review paper were to summarize the available serological and molecular assays for the diagnosis of SRLVs, to highlight their diagnostic performance emphasizing on advantages and drawbacks of their application, and to discuss current and future perspectives, challenges, limitations and impacts regarding the development of reliable and efficient tools for the diagnosis of SRLVs infections.

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“…Tables derived from GISAID were transformed with dplyr package in R Studio and the results were visualized with Microsoft Office Excel, in order to assess the nucleotide substitutions found on primer hybridization regions. Further analysis was performed in silico using the DINAMelt software [14] to estimate, at the annealing temperature of 58 • C, the mole fraction of each primer hybridized to the target sequences with most common single mismatches in SARS-CoV-2 genomic sequences (frequency > 4%) [15]. Four LNA TaqMan probes were used, each conjugated with a different fluorophore at the 5' end (FAM, HEX, Texas Red and Cy5) for fluorescence signal discrimination (Table 1).…”

Section: Primers Taqman Probes and Non-extendable Blocking Oligonucleotidesmentioning

confidence: 99%

A Novel Real-Time RT-PCR-Based Methodology for the Preliminary Typing of SARS-CoV-2 Variants, Employing Non-Extendable LNA Oligonucleotides and Three Signature Mutations at the Spike Protein Receptor-Binding Domain

Chaintoutis

Chassalevris

Balaska

et al. 2021

Life

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Mutations resulting in amino-acid substitutions of the SARS-CoV-2 spike protein receptor-binding domain (RBD) have been associated with enhanced transmissibility and immune escape of the respective variants, namely Alpha, Beta, Gamma or Delta. Rapid identification of the aforementioned variants of concern and their discrimination of other variants is thus of importance for public health interventions. For this reason, a one-step real-time RT-PCR assay employing four locked nucleic acid (LNA) modified TaqMan probes was developed, to target signature mutations associated with amino-acid substitutions at positions 478, 484 and 501 present in the receptor-binding motif (RBM) of the spike protein RBD. This region contains most contacting residues of SARS-CoV-2 that bind to ACE2. A novel strategy employing the use of non-extendable LNA oligonucleotide blockers that can reduce non-specific hybridization of probes increased the number of different mutated sites examined in a multiplex PCR. The combinatory analysis of the different fluorescence signals obtained enabled the preliminary differentiation of SARS-CoV-2 variants of concern. The assay is sensitive with a LOD of 263 copies/reaction for the Delta variant, 170 copies/reaction for the Beta variant, amplification efficiencies > 91% and a linear range of >5 log10 copies/reaction against all targets. Validation of the assay using known SARS-CoV-2-positive and negative samples from humans and animals revealed its ability to correctly identify the targeted mutations and preliminary characterize the SARS-CoV-2 variants. The novel approach for mutation typing using LNA oligonucleotide blockers can be modified to target signature mutations at four different sites in the RBM and further expand the range of variants detected.

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A highly sensitive semi-nested real-time PCR utilizing oligospermine-conjugated degenerate primers for the detection of diverse strains of small ruminant lentiviruses

Cited by 18 publications

References 65 publications

Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping

Accurate Diagnosis of Small Ruminant Lentivirus Infection Is Needed for Selection of Resistant Sheep through TMEM154 E35K Genotyping

Serological, Molecular and Culture-Based Diagnosis of Lentiviral Infections in Small Ruminants

A Novel Real-Time RT-PCR-Based Methodology for the Preliminary Typing of SARS-CoV-2 Variants, Employing Non-Extendable LNA Oligonucleotides and Three Signature Mutations at the Spike Protein Receptor-Binding Domain

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