Nucleotide amplification and sequencing of the GC-rich region between matrix and fusion protein genes of peste des petits ruminants virus

Kinimi, Edson; Hakizimana, J.N.; Misinzo, Gerald

doi:10.1016/j.jviromet.2021.114390

Cited by 2 publications

(3 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, amplifying the high GC content of DNA fragments is more difficult than for nonhigh GC content target fragments [1]. GC-rich sequences in the human genome are common and include important regulatory domains such as promoters, boosters, and control elements [2,3]. PCR amplification of GC-rich DNA is often problematic because the secondary structure of DNA is stable and not easy to melt.…”

Section: Introductionmentioning

confidence: 99%

“…The mechanism of nano-PCR is still unclear because of the PCR reaction system's complexity and materials' characteristics. The possible mechanisms are as follows: (1) surface interactions between materials and PCR components [19][20][21][22][23]; (2) thermal conversion of materials rate [24,25]; (3) electrostatic interaction [26][27][28]; (4) analogous to ssDNA binding protein (SSB) [22]; (5) catalytic activity [29]. There is no doubt that these mechanisms cannot explain the impact on all the materials in PCR, and more undiscovered mechanisms need to be explored.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Enhancement Effects and Mechanism Studies of Two Bismuth-Based Materials Assisted by DMSO and Glycerol in GC-Rich PCR

et al. 2023

View full text Add to dashboard Cite

Polymerase chain reaction (PCR) has extensive bioanalytical applications in molecular diagnostics and genomic research studies for rapid detection and precise genomic amplification. Routine integrations for analytical workflow indicate certain limitations, including low specificity, efficiency, and sensitivity in conventional PCR, particularly towards amplifying high guanine–cytosine (GC) content. Further, there are many ways to enhance the reaction, for example, using different PCR strategies such as hot-start/touchdown PCR or adding some special modifications or additives such as organic solvents or compatible solutes, which can improve PCR yield. Due to the widespread use of bismuth-based materials in biomedicine, which have not yet been used for PCR optimization, this attracts our attention. In this study, two bismuth-based materials that are inexpensive and readily available were used to optimize GC-rich PCR. The results demonstrated that ammonium bismuth citrate and bismuth subcarbonate effectively enhanced PCR amplification of the GNAS1 promoter region (∼84% GC) and APOE (75.5% GC) gene of Homo sapiens mediated by Ex Taq DNA polymerase within the appropriate concentration range. Combining DMSO and glycerol additives was critical in obtaining the target amplicons. Thus, the solvents mixed with 3% DMSO and 5% glycerol were used in bismuth-based materials. That allowed for better dispersion of bismuth subcarbonate. As for the enhanced mechanisms, the surface interaction of PCR components, including Taq polymerase, primer, and products with bismuth-based materials, was maybe the main reason. The addition of materials can reduce the melting temperature (Tm), adsorb polymerase and modulate the amount of active polymerase in PCR, facilize the dissociation of DNA products, and enhance the specificity and efficiency of PCR. This work provided a class of candidate enhancers for PCR, deepened our understanding of the enhancement mechanisms of PCR, and also explored a new application field for bismuth-based materials.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enhancement Effects and Mechanism Studies of Two Bismuth-Based Materials Assisted by DMSO and Glycerol in GC-Rich PCR

et al. 2023

View full text Add to dashboard Cite

show abstract

“…Exposure to PPRV is primarily diagnosed by several serological assays that target anti-PPRV antibodies [3]. The genome of the virus is composed of six genes in the order of 3'-N, P(C/V), M, F, H, and L-5' [4]. The encoded proteins, which include haemagglutinin protein (H), large polymerase protein (L), fusion protein (F), matrix protein (M), and phosphoprotein (P), all contain the individual genes from which they were produced [5].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of diagnostic performance of H-based blocking ELISA for specific detection of peste des petits ruminants in domestic sheep, goats, cattle and camels

Lelisa¹,

Chibssa²,

Desissa

et al. 2022

BMC Microbiol

View full text Add to dashboard Cite

Introduction Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats, peste des petits ruminants (PPR), which is targeted for global control and eradication by 2030. The serological diagnostic tool kits for accurate diagnosis of PPR have inherent strengths and weaknesses that require parallel validation and optimization across animal species. Thus, the objective of this study was to evaluate diagnostic performance of haemagglutinin based PPR blocking ELISA (HPPR- b-ELISA), that was developed by Africa Union Pan African Veterinary Vaccine Center for specific detection of anti- PPRV antibodies. Methods In preliminarily investigation, diagnostic performance of the HPPR-b-ELISA®, commercial PPR competition ELISA (c-ELISA) and virus neutralization test (VNT) were compared for the detection of anti-PPRV antibodies in goats, sheep, cattle and camels. Results The sensitivity and specificity of HPPR- b-ELISA® were 79.55 and 99.74%, respectively, compared to c-ELISA. The HPPR- b-ELISA® was in perfect agreement (κ = 0.86) with the c-ELISA in all sera collected from goats, sheep and cattle. There was almost perfect agreement between the species of goats (κ = 0.82) and sheep (κ = 0.98), while the agreement was substantial in cattle (κ = 0.78) and no agreement was observed in camels (κ = 0.00). Similarly, the sensitivity and specificity of the HPPR b-ELISA were 80 and 96.36%, respectively compared to VNT with almost perfect agreement in goats (κ = 0.83) and sheep (κ = 0.89), moderate in cattle (κ = 0.50) and none in camels (κ = 0.00). Conclusion Our study revealed that HPPR- b-ELISA is a suitable and valid method that can alternatively be used for screening and monitoring of PPR in sheep, goats and cattle except for camels.

show abstract

Nucleotide amplification and sequencing of the GC-rich region between matrix and fusion protein genes of peste des petits ruminants virus

Cited by 2 publications

References 43 publications

Enhancement Effects and Mechanism Studies of Two Bismuth-Based Materials Assisted by DMSO and Glycerol in GC-Rich PCR

Enhancement Effects and Mechanism Studies of Two Bismuth-Based Materials Assisted by DMSO and Glycerol in GC-Rich PCR

Evaluation of diagnostic performance of H-based blocking ELISA for specific detection of peste des petits ruminants in domestic sheep, goats, cattle and camels

Contact Info

Product

Resources

About