Specific PCR and real-time PCR assays for detection and quantitation of ‘Candidatus Phytoplasma phoenicium’

Jawhari, M.; Abrahamian, Peter; Sater, Ali Abdel; Sobh, H.; Tawidian, Patil; Abou‐Jawdah, Y.

doi:10.1016/j.mcp.2014.12.003

Cited by 18 publications

(9 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The extension temperature of 60-68 • C (Step 4) is recommended for regular PCR. There were no differences found in experiments using "touch-down" annealing temperatures reduced by 0.6-1.0 • C per cycle as recommended in the KASP method (Jatayev et al, 2017). Therefore, a fixed annealing temperature was used in the first round of amplification.…”

Section: Modified Pcr Protocol For Snp Genotypingmentioning

confidence: 99%

“…Such systems generally have two essential components: (1) allele-specific primers or molecular probe targeting the SNP and (2) a system of dye and quencher with at least one donor that produces a fluorescent signal and an acceptor that quenches the fluorescence when in close proximity (Chen and Sullivan, 2003 ; Giancola et al, 2006 ; Mamotte, 2006 ). In some methods, like Scorpions (Thelwell et al, 2000 ; Solinas et al, 2001 ) TaqMan (Schena et al, 2006 ; Jawhari et al, 2015 ) and Molecular Beacons (Hardinge and Murray, 2019 ), these two parts are combined in a single molecular probe.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

et al. 2022

View full text Add to dashboard Cite

The proposed method is a modified and improved version of the existing “Allele-specific q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.

show abstract

Section: Modified Pcr Protocol For Snp Genotypingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

et al. 2022

View full text Add to dashboard Cite

show abstract

“…‘ Ca . P. phoenicium’ can be found in flower petals, leaf petioles or midribs, but the highest concentration is in the phloem tissue of stems and roots (Jawhari et al, 2015). In Lebanon, ‘ Ca .…”

Section: Detectionmentioning

confidence: 99%

PM 7/150 (1) ‘Candidatus Phytoplasma phoenicium’

2021

EPPO Bulletin

View full text Add to dashboard Cite

show abstract

“…Black hole quenchers, BHQ1, BHQ2 and BHQ3, were developed and used in plant pathology with TaqMan PCR [ 21 , 22 ] and in medical research [ 23 ]. Only BHQ1 has the maximum absorbance in the spectra of all fluorophores, FAM, JOE, VIC and HEX (Additional file 1 ).…”

Section: Introductionmentioning

confidence: 99%

Advantages of Amplifluor-like SNP markers over KASP in plant genotyping

et al. 2017

View full text Add to dashboard Cite

BackgroundKASP (KBioscience Competitive Allele Specific PCR) and Amplifluor (Amplification with fluorescence) SNP markers are two prominent technologies based upon a shared identical Allele-specific PCR platform.MethodsAmplifluor-like SNP and KASP analysis was carried out using published and own design of Universal probes (UPs) and Gene-specific primers (GSPs).ResultsAdvantages of the Amplifluor-like system over KASP include the significantly lower costs and much greater flexibility in the adjustment and development of ‘self-designed’ dual fluorescently-labelled UPs and regular GSPs. The presented results include optimisation of ‘tail’ length in UPs and GSPs, protocol adjustment, and the use of various fluorophores in different qPCR instruments. The compatibility of the KASP Master-mix in both original and Amplifluor-like systems has been demonstrated in the presented results, proving their similar principles. Results of SNP scoring with rare alleles in addition to more frequently occurring alleles are shown.ConclusionsThe Amplifluor-like system produces SNP genotyping results with a level of sensitivity and accuracy comparable to KASP but at a significantly cheaper cost and with much greater flexibility for UPs with self-designed GSPs.Electronic supplementary materialThe online version of this article (10.1186/s12870-017-1197-x) contains supplementary material, which is available to authorized users.

show abstract

Specific PCR and real-time PCR assays for detection and quantitation of ‘Candidatus Phytoplasma phoenicium’

Cited by 18 publications

References 29 publications

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

Modified “Allele-Specific qPCR” Method for SNP Genotyping Based on FRET

PM 7/150 (1) ‘Candidatus Phytoplasma phoenicium’

Advantages of Amplifluor-like SNP markers over KASP in plant genotyping

Contact Info

Product

Resources

About