scholarly journals Comparison of Direct Sequencing with Real-time PCR High Resolution Melt and PCR Restriction Fragment Length Polymorphism Analysis to Identify Clinically Important Candida Species

2021 ◽  
Vol 16 (4) ◽  
Author(s):  
Zeynab Yassin ◽  
Fariba Shirvani ◽  
Mahsa Fattahi
Keyword(s):  
Polymerase Chain Reaction ◽  
High Resolution ◽  
Real Time ◽  
Fragment Length ◽  
Sanger Sequencing ◽  
Chain Reaction ◽  
High Resolution Melt ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Restriction Fragment Length

Background: Candida albicans is the predominant yeast reported from human infection. Non-albicans Candida species have been recently developed as medically vital fungi. Therefore, it is essential to detect and identify the pathogens at the species level to prescribe appropriate treatment. Methods: This study assessed two complementary methods, including real-time polymerase chain reaction-high resolution melt (PCR-HRM) and polymerase chain reaction-restriction fragment length morphism (PCR-RFLP) with standard PCR and Sanger sequencing as the benchmark. Results: In total, 66 samples were tested, and two newly-advanced assays were more effective and displayed comprehensive concordance (66/66, 100%) with Sanger sequencing outcomes. Moreover, accurate and economical tests were positively advanced by real-time PCR-HRM for C. albicans and C. parapsilosis complexes. Conclusions: Given the number of studies performed on the comparison of sensitivity and specificity of phenotypic and genotypic methods to diagnose and identify invasive fungal pathogens and the findings of this study, it could be stated that the correlative PCR-HRM and PCR-RFLP methods were effectively advanced as substitutes for conventional Sanger sequencing for the reasonable identification. However, supplementary evaluations and confirming studies should be carried out with a broad range of samples to standardize this method for routine application in medical laboratories.

scholarly journals Análise de polimorfismo do gene da somatotropina em vacas Nelore e seu efeito sobre o peso à desmama de suas progênies

1999 ◽  
Vol 51 (6) ◽  
pp. 565-570
Author(s):  
F.J.C. Faria ◽  
S.E.F. Guimarães ◽  
R.M.G. Lima ◽  
G.B. Mourão ◽  
L.E.L. Pinheiro
Keyword(s):  
Polymerase Chain Reaction ◽  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Chain Reaction ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Restriction Fragment Length

Informações sobre peso à desmama de um rebanho Nelore foram utilizadas após ajuste para idade padrão de 205 dias, sexo da cria, idade da mãe, touro e mês de desmama, para separar as reprodutrizes em dois grupos, cujos filhos diferiam nesse peso. As médias ajustadas pelo método dos quadrados mínimos foram para os grupos pesado (P) e leve (L) de 163,21± 2,18kg e 134,44± 2,18kg, respectivamente, com 41 animais em cada grupo. Essas reprodutrizes foram submetidas à coleta de sangue para estudo de polimorfismos do gene da somatotropina bovina, pela técnica de PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism). A amplificação de uma região entre o éxon III e V do gene da somatotropina permitiu analisar dois sítios de restrição. Para o sítio do éxon V, todos os animais foram identificados como monomórficos (Leu-Leu). Quanto ao sítio do íntron 3, foi possível identificar os seguintes genótipos 21 (+/-) e 60 (-/-), com as freqüências de 0,13 e 0,87 para os alelos (+) e (-), respectivamente. O peso dos filhos dos animais com o genótipo +/- foi de 152,42± 4,41kg e os -/- 147,60± 2,61kg. Os grupos P e L não diferiram entre si quanto às freqüências alélicas apresentadas. O genótipo das reprodutrizes não afetou o peso à desmama de suas crias, existindo portanto outros efeitos genéticos e não genéticos de maior magnitude.

scholarly journals Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Authentication of Raw Meats from Game Birds

2008 ◽  
Vol 91 (6) ◽  
pp. 1416-1422 ◽  
Author(s):  
María Rojas ◽  
Isabel González ◽  
Violeta Fajardo ◽  
Irene Martín ◽  
Pablo E Hernández ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Chain Reaction ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Restriction Fragment Length

Abstract Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis has been applied to the identification of meats from quail (Coturnix coturnix), pheasant (Phasianus colchicus), red-legged partridge (Alectoris rufa), guinea fowl (Numida meleagris), capercaillie (Tetrao urogallus), Eurasian woodcock (Scolopax rusticola), woodpigeon (Columba palumbus), and song thrush (Turdus philomelos). PCR amplification was performed using a set of primers flanking a conserved region of approximately 720 base pairs (bp) from the mitochondrial 12S rRNA gene. Restriction site analysis based on sequence data from this DNA fragment permitted the selection of Alul and BfaI endonucleases for species identification. The restriction profiles obtained when amplicons were digested with the chosen enzymes allowed the unequivocal identification of all game bird species analyzed. However, the use of the PCR-RFLP technique described is limited to raw meat authentication. It is not suitable for cooked products because thermal treatment strongly accelerates DNA degradation leading to difficulties in amplifying the 720 bp fragment.

Species identification in seven small millet species using polymerase chain reaction - restriction fragment length polymorphism of trnS-psbC gene region

Genome ◽  
2001 ◽  
Vol 44 (3) ◽  
pp. 495-499 ◽  
Author(s):  
M Parani ◽  
K Rajesh ◽  
M Lakshmi ◽  
L Parducci ◽  
A E Szmidt ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Chain Reaction ◽  
Pcr Rflp ◽  
Enzyme Combination ◽  
Polymerase Chain ◽  
Restriction Fragment Length

The chloroplast trnS-psbC gene regions from total genomic DNA of 119 accessions from seven small millet species were amplified by polymerase chain reaction (PCR) and digested with eight restriction enzymes individually as well as in combinations of two enzymes to generate restriction fragment length polymorphism (PCR–RFLP). PCR–RFLP with individual enzymes revealed polymorphism between only some species. However, all the species could be distinguished by using a combination of two enzymes, specifically HaeIII and MspI. PCR–RFLP of 11 to 20 accessions with the same enzyme combination showed no intraspecific variation, which established that the differential banding patterns were species specific. In contrast, the same enzyme combination was not useful for differentiating different species of the genera Cajanus, Rhyncosia, Abies, Rhizophora, Ceriops, and Bruguiera, and it also revealed intraspecies variation in three species of Abies. The present study indicated that digestion of trnS-psbC with two four-base recognizing enzymes reveals more variation than with either enzyme alone and that it may be a method of choice for species identification in some genera.Key words: small millets, PCR–RFLP, trnS-psbC, chloroplast DNA.

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