scholarly journals Development of a PCR-restriction fragment length polymorphism assay for detection and subtyping of cholix toxin variant genes of Vibrio cholerae

2014 ◽  
Vol 63 (5) ◽  
pp. 667-673 ◽  
Author(s):  
Sharda Prasad Awasthi ◽  
Masahiro Asakura ◽  
Sucharit Basu Neogi ◽  
Atsushi Hinenoya ◽  
T. Ramamurthy ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Vibrio Cholerae ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Pcr Rflp ◽  
Cholix Toxin ◽  
Rflp Assay ◽  
Restriction Fragment Length

Cholix toxin (ChxA) is an exotoxin reported in Vibrio cholerae non-O1/non-O139. Apart from its prototype (ChxA I) we have recently identified two novel variants of this toxin, ChxA II and ChxA III. Our previous investigations indicated that the first two variants may instigate extra-intestinal infections and ChxA II can be more lethal than ChxA I in mice. However, all three cholix toxins (ChxA I to III) failed to show any enterotoxicity in rabbit ileal loops. In this study we developed a PCR-restriction fragment length polymorphism (RFLP) assay to differentiate all three chxA variants to further understand the importance of each subtype. By using 53 V. cholerae non-O1/non-O139 strains harbouring chxA genes, which were previously categorized by sequencing, and various other strains as negative controls, the PCR-RFLP assay showed 100 % typability and specificity. Furthermore, when applied to differentiate additional V. cholerae strains, which were also screened for the chxA gene by colony hybridization, this assay identified chxA I and chxA II genes among 18.5 % and 4.5 % of non-O1/non-O139 strains (n = 178), respectively. One non-O1/non-O139 strain was untypable due to the insertion of an IS911-like element. Interestingly, the chxA I gene was detected in 10 out of 137 cholera toxin gene-negative V. cholerae O1 strains. These results suggest that the PCR-RFLP assay developed in this study can be a rapid and simple method to differentiate the chxA subtypes.

scholarly journals Το κληρονομικό αγγειοοίδημα στην Ελλάδα και ο ρόλος των γενετικών βλαβών γονιδίων που κωδικοποιούν ένζυμα της οδού καλλικρεϊνης -βραδυκινίνης στην κλινική έκφρασή του

2016 ◽  
Author(s):  
Νικόλαος Κουτσοστάθης
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Pcr Rflp ◽  
Restriction Fragment Length

ΣΚΟΠΟΣ Η παρουσίαση των πρώτων αποτελεσμάτων της πανελλήνιας καταγραφής ασθενών με κληρονομικό αγγειοοίδημα (ΚΑΟ) κατά την τελευταία τετραετία (Ιούλιος 2010 – Ιούνιος 2014) και η αναζήτηση της συσχέτισης των πολυμορφισμών γονιδίων, τα οποία παράγουν πρωτεΐνες της οδού του συστήματος κινινών- καλλικρεΐνης, με φαινοτυπικά χαρακτηριστικά του ΚΑΟ.ΥΛΙΚΟ-ΜΕΘΟΔΟΙ Έγινε πανελλήνια συστηματική καταγραφή των περιπτώσεων ΚΑΟ μέσω ευρείας συνεργασίας με ιατρούς και νοσοκομεία σε όλη τη χώρα. Σε όσους ασθενείς συμμετείχαν στην μελέτη συμπληρώθηκε: α) τυποποιημένο ερωτηματολόγιο για την καταγραφή δημογραφικών - κλινικών και θεραπευτικών χαρακτηριστικών της νόσου και β) γενεαλογικό δέντρο σε κάθε οικογένεια. Σε κάθε συμμετέχοντα ασθενή έγινε λήψη περιφερικού αίματος για τον προσδιορισμό των πολυμορφισμών F12-5046T>C (rs1801020), BDKRB1-(-699G>C) (rs4905475), SERPING1-21963G>A (p.V480M, rs4926) και ACE I/D (rs1799752). Η ανίχνευση των πολυμορφισμών έγινε με την τεχνική PCR-RFLP (restriction fragment length polymorphism), δηλ. μέσω ενίσχυσης τμημάτων DNA με PCR και εν συνεχεία πέψης με ένζυμα περιορισμού για τους τρεις πρώτους και με απλή PCR για τον τελευταίο. Σε ορισμένα δείγματα χρησιμοποιήθηκε ανάλυση αλληλουχίας βάσεων για την επιβεβαίωση των αποτελεσμάτων. Ως μάρτυρες χρησιμοποιήθηκαν δείγματα περιφερικού αίματος από υγιείς δότες. ΑΠΟΤΕΛΕΣΜΑΤΑ Καταγράφηκαν 128 ασθενείς με ΚΑΟ τύπου Ι και ΙΙ που ανήκουν σε 42 μη συγγενείς οικογένειες (επιπολασμός της νόσου στην Ελλάδα 1:84.000). Επιπρόσθετα καταγράφηκε και μια οικογένεια με 3 μέλη με ΚΑΟ με φυσιολογικό C1-INH. Σε 85 από τους ως άνω ασθενείς, από 34 οικογένειες, έγινε γενετική ανάλυση. Η μέση καθυστέρηση της διάγνωσης της νόσου ήταν τα 17.5 έτη (εύρος 0-58 έτη) , αλλά η διάγνωση ασθενών με ΚΑΟ έχει σαφώς αυξηθεί την τελευταία δεκαετία. 16.7% των ασθενών είχαν υποβληθεί σε διασωλήνωση της τραχείας ή τραχειοτομή λόγω οιδήματος λάρυγγα και 23.5% είχαν υποβληθεί σε άσκοπες χειρουργικές επεμβάσεις λόγω κρίσεων αγγειοοιδήματος στο πεπτικό. Η συχνότητα των θανάτων λόγω ΚΑΟ ήταν περίπου 1 ανά 3 οικογένειες και αφορούσε στην πλειοψηφία τους μη διαγνωσμένους ασθενείς. Το 10.4% των ασθενών δήλωσαν ότι δεν είχε οποιοδήποτε φάρμακο για την αντιμετώπιση των επεισοδίων ΑΟ και 84.7% ανέφεραν ότι η ζωή τους ήταν επηρεασμένη από μέτρια ως σοβαρά λόγω της νόσου. Η παρουσία του πολυμορφισμού F12-5046T>C και του BDKRB1-(-699G>C) καθυστερούσε την έναρξη του ΚΑΟ κατά 4.6 έτη (p=0.03) και κατά 9.4 έτη (p=0.02) αντίστοιχα. Στους ομόζυγους ή ετερόζυγους με πολυμορφισμό V480M στο SERPING1 κυριαρχούσαν οι κρίσεις από το δέρμα. ΣΥΜΠΕΡΑΣΜΑΤΑ Η παρούσα διατριβή, η οποία αποτέλεσε την πρώτη προσπάθεια πανελλαδικής καταγραφής ασθενών με ΚΑΟ, κατάφερε να καλύψει το μεγαλύτερο τμήμα της χώρας. Στην Ελλάδα υπήρχε σημαντική καθυστέρηση της διάγνωσης του ΚΑΟ, από τις μεγαλύτερες παγκοσμίως, η οποία την τελευταία δεκαετία φαίνεται να αναστρέφεται θεαματικά. Το γεγονός αυτό σε συνδυασμό με την θεραπευτική αντιμετώπιση, η οποία σαφώς υπολείπεται της διεθνούς πραγματικότητας, έχουν δυσμενείς συνέπειες για τους ασθενείς. Έτσι αναδεικνύεται η ανάγκη για την προώθηση πρακτικών αντιμετώπισης του προβλήματος. Τα υπόλοιπα επιδημιολογικά και κλινικά χαρακτηριστικά του ΚΑΟ στη χώρα μας δε διαφέρουν από τα αναφερόμενα διεθνώς. Οι πολυμορφισμοί F12-5046T>C και του BDKRB1-(-699G>C) σχετίζονται σημαντικά με καθυστερημένη έναρξη της νόσου και άρα ηπιότερη νόσηση από ΚΑΟ, ενώ ο SERPING1-21963G>A με εντόπιση κρίσεων αγγειοοιδήματος στο δέρμα. Σε πολύ πρόσφατη πολυκεντρική Ευρωπαϊκή μελέτη, προέκταση της παρούσας μελέτης, επιβεβαιώθηκε ότι ο πολυμορφισμός F12-5046T>C αποτελεί τον μοναδικό, μέχρι στιγμής, ανεξάρτητο γενετικό παράγοντα με τόσο ισχυρή επίδραση στον φαινότυπο και την πρόγνωση της νόσου.

scholarly journals Identification of Pneumococcal Serotypes by PCR–Restriction Fragment Length Polymorphism

Diagnostics ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 196 ◽  
Author(s):  
García-Suárez ◽  
González-Rodríguez ◽  
Cima-Cabal ◽  
Yuste ◽  
Vazquez ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Dna Sequences ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Restriction Enzymes ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Streptococcus pneumoniae shows more than 90 capsular serotypes that can be distinguished by their reactivity against antisera. The main objective of this work was the development of a molecular method for serotyping without the use of antisera. A computer program containing an algorithm was used to search in a database for potentially useful enzymes for Restriction Fragment Length Polymorphism-RFLP typing, in order to maximize the discrimination between different serotypes. DNA sequences of 90 serotypes for the region between dexB and aliA genes were compiled, and a computer screening of restriction enzymes was performed. The wzg–wzh–wzd–wze region and Sse9I restriction predicted unique PCR-RFLP patterns for 39 serotypes and eight serogroups. A second restriction enzyme resolved fragment specific patterns for 25 serotypes. The method was tested with 98 serotype-unknown clinical isolates. PCR-RFLP analysis deduced correct serotypes that were confirmed by Quellung reaction for 78.5% of the isolates.

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.

scholarly journals Rapid Identification of Listeria Species by Using Restriction Fragment Length Polymorphism of PCR-Amplified 23S rRNA Gene Fragments

2003 ◽  
Vol 69 (11) ◽  
pp. 6386-6392 ◽  
Author(s):  
Delphine Paillard ◽  
Véronique Dubois ◽  
Robert Duran ◽  
Fany Nathier ◽  
Catherine Guittet ◽  
...  
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
23S Rrna ◽  
Rrna Gene ◽  
Pcr Rflp ◽  
23S Rrna Gene ◽  
Restriction Fragment Length

ABSTRACT A molecular method based on restriction fragment length polymorphism (RFLP) of PCR-amplified fragments of the 23S rRNA gene was designed to rapidly identify Listeria strains to the species level. Two fragments (S1, 460 bp, and S2, 890 bp) were amplified from boiled DNA. S2 was cut with the restriction enzymes XmnI or CfoI and, if needed, S1 was digested by either AluI or ClaI. This method was first optimized with six reference strains and then applied to 182 isolates collected from effluents of treatment plants. All isolates were also identified by the API Listeria kit, hemolysis, and phosphatidylinositol-specific phospholipase C production (PI-PLC) on ALOA medium. The PCR-RFLP method unambiguously identified 160 environmental strains, including 131 in concordance with the API system, and revealed that 22 isolates were mixed cultures of Listeria monocytogenes and Listeria innocua. Discrepant results were resolved by a multiplex PCR on the iap gene, which confirmed the PCR-RFLP data for 49 of the 51 discordances, including the 22 mixed cultures. Sequencing of the 16S rRNA gene for 12 selected strains and reconstruction of a phylogenetic tree validated the molecular methods, except for two unclassifiable strains. The 158 single identifiable isolates were 92 L. monocytogenes (including seven nonhemolytic and PI-PLC-negative strains), 61 L. innocua, 4 Listeria seeligeri, and 1 Listeria welshimeri strain. The PCR-RFLP method proposed here provides rapid, easy-to-use, inexpensive, and reliable identification of the six Listeria species. Moreover, it can detect mixtures of Listeria species and thus is particularly adapted to environmental and food microbiology.

scholarly journals Molecular detection of Candida species by Restriction Fragment Length Polymorphism (RFLP) analysis of PCR from HIV infected persons

2019 ◽  
Vol 10 (3) ◽  
pp. 1596-1601
Author(s):  
SandhyaRani T ◽  
Srikumar R ◽  
Prabhakar Reddy E ◽  
Latha S
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Candida Species ◽  
Fragment Length Polymorphism ◽  
Oral Candidiasis ◽  
Candida Spp ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Oral Candidiasis an important opportunistic fungal infection in HIV patients. This is the first sign of HIV infection. It can spread from the mouth through the pharynx to the oesophagus. Oral Candidiasis is most regularly complicated with oesophagal Candidiasis, which may restrain nourishment and result in weight reduction, alarming the overall health of the HIV tainted persons. In present study, we have a tendency to determine in Candida species by blend of Restriction Fragment Length Polymorphism (RFLP) examination of PCR from HIV tainted people and furthermore we demonstrated that phenotypic and genotypic identification method was vital to work out the species. We recognized seventy two Candida confines acquired from a hundred and fifty patients contaminated with HIV by utilizing PCR-RFLP assay. All inclusive primers for the internal transcribed spacer (ITS) area (ITS1– ITS4) of the fungal DNA qualities were utilized for this measure. We distinguished the diverse Candida spp. By utilizing the limitation enzyme MspI. Here most of the isolates were obtained from male patients 40(51%). Candida albicans was the recurrent species segregated (46.8%) pursued by C. tropicalis (30.3%), C. glabrata (7.59%), C. parapsilosis (6.3%) C.dubulinesis (6.3%) along with C. krusei (2.5%) detected successfully. PCR- RFLP is a high definite, sensitive, quick, reliable technique and also applicable method for fungal detection in clinical laboratory for identification of medically important Candida spp. It is used for detection of mycological examinations in HIV-positive along with other immunocompromised patients for epidemiological studies.

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