Detection of Cryptosporidium in stool sample by PCR-RFLP v1

2021 ◽  
Author(s):  
botchiesenyo not provided
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Mineral Waters ◽  
Natural Mineral ◽  
Cryptosporidium Spp ◽  
Drinking Waters ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Nested PCR-RFLP adapted from Nichols, R.A.B., Campbell, B.M. and Smith, H.V., 2003. Identification of Cryptosporidium spp. oocysts in United Kingdom noncarbonated natural mineral waters and drinking waters by using a modified nested PCR-restriction fragment length polymorphism assay. Applied and environmental microbiology, 69(7), p.4183.

scholarly journals Identification of Cryptosporidium spp. Oocysts in United Kingdom Noncarbonated Natural Mineral Waters and Drinking Waters by Using a Modified Nested PCR-Restriction Fragment Length Polymorphism Assay

2003 ◽  
Vol 69 (7) ◽  
pp. 4183-4189 ◽  
Author(s):  
R. A. B. Nichols ◽  
B. M. Campbell ◽  
H. V. Smith
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Water Samples ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Mineral Waters ◽  
Natural Mineral ◽  
Drinking Waters ◽  
Restriction Fragment Length

ABSTRACT We describe a nested PCR-restriction fragment length polymorphism (RFLP) method for detecting low densities of Cryptosporidium spp. oocysts in natural mineral waters and drinking waters. Oocysts were recovered from seeded 1-liter volumes of mineral water by filtration through polycarbonate membranes and from drinking waters by filtration, immunomagnetizable separation, and filter entrapment, followed by direct extraction of DNA. The DNA was released from polycarbonate filter-entrapped oocysts by disruption in lysis buffer by using 15 cycles of freeze-thawing (1 min in liquid nitrogen and 1 min at 65°C), followed by proteinase K digestion. Amplicons were readily detected from two to five intact oocysts on ethidium bromide-stained gels. DNA extracted from Cryptosporidium parvum oocysts, C. muris (RN 66), C. baileyi (Belgium strain, LB 19), human-derived C. meleagridis, C. felis (DNA from oocysts isolated from a cat), and C. andersoni was used to demonstrate species identity by PCR-RFLP after simultaneous digestion with the restriction enzymes DraI and VspI. Discrimination between C. andersoni and C. muris isolates was confirmed by a separate, subsequent digestion with DdeI. Of 14 drinking water samples tested, 12 were found to be positive by microscopy, 8 were found to be positive by direct PCR, and 14 were found to be positive by using a nested PCR. The Cryptosporidium species detected in these finished water samples was C. parvum genotype 1. This method consistently and routinely detected >5 oocysts per sample.

scholarly journals Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning

2011 ◽  
Vol 77 (12) ◽  
pp. 3998-4007 ◽  
Author(s):  
Norma J. Ruecker ◽  
Rebecca M. Hoffman ◽  
Rachel M. Chalmers ◽  
Norman F. Neumann
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Nested Pcr ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Content Type ◽  
Pcr Rflp ◽  
Pcr Products ◽  
Restriction Fragment Length

ABSTRACTMolecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene ofCryptosporidiumspecies were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species (Cryptosporidium hominis,C. parvum,C. felis,C. meleagridis,C. ubiquitum,C. muris, andC. andersoni) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies forC. andersoniandC. parvum, respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures ofCryptosporidiumat template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity ofCryptosporidium, particularly for environmental water samples, in which mixtures of parasites are common.

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 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.

PCR–Restriction Fragment Length Polymorphism and Pulsed-Field Gel Electrophoresis Characterization of Listeria monocytogenes Isolates from Ready-to-Eat Foods, the Food Processing Environment, and Clinical Samples in South Africa

2020 ◽  
Vol 83 (3) ◽  
pp. 518-533
Author(s):  
DIANE RIP ◽  
PIETER A. GOUWS
Keyword(s):  
South Africa ◽  
Listeria Monocytogenes ◽  
Restriction Fragment Length Polymorphism ◽  
Length Polymorphism ◽  
Food Processing ◽  
Fragment Length Polymorphism ◽  
Rflp Analysis ◽  
Pcr Rflp ◽  
Restriction Fragment Length ◽  
Food Processing Environment

ABSTRACT Listeria monocytogenes is a ubiquitous, intracellular foodborne pathogen that is responsible for invasive listeriosis. The ability of L. monocytogenes to cause disease has some correlation with the serotypes of a specific lineage group, making the identification of lineage groups important for epidemiological analysis. The development of typing methods to link the strains of L. monocytogenes to an outbreak of listeriosis would help minimize the spread of the disease. The aim of this study was to design a PCR–restriction fragment length polymorphism (RFLP) method to differentiate between the lineage groups of L. monocytogenes. PCR-amplified fragments of the hly gene for 12 serotypes of L. monocytogenes were sequenced, aligned, and analyzed with the BioEdit program, and single nucleotide polymorphisms (SNPs) within regions of this gene were identified. Because of the difficulty in acquiring a serotype 4ab reference strain, this serotype was not included in this study. We tested the specificity and accuracy of the PCR-RFLP method on these L. monocytogenes reference strains and validated the method with 172 L. monocytogenes strains recovered from humans, food, and the food processing environment in 2000 to 2002 and 2008 to 2010 from regions within South Africa. PCR-RFLP analysis applied in this study placed L. monocytogenes serotypes into one of three lineage groups based on the sequence differences and SNPs within each lineage group. The SNPs were conserved in a region where RFLP analysis could be applied for a distinction between L. monocytogenes lineage groups. HIGHLIGHTS

scholarly journals Restriction fragment length polymorphism analysis of genes of virulent strain isolate of Toxoplasma gondii using enzyme DdeI

2021 ◽  
pp. 196-203
Author(s):  
Fitrine Ekawasti ◽  
Umi Cahyaningsih ◽  
N. L. P. Indi Dharmayanti ◽  
Siti Sa'diah ◽  
Didik Tulus Subekti ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Restriction Fragment Length Polymorphism ◽  
Genetic Markers ◽  
Restriction Enzyme ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Virulent Strain ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Background and Aim: Toxoplasma gondii is a unicellular coccidian parasite distributed globally and is an important zoonotic pathogen. Approximately 30% of the human population worldwide is chronically infected with T. gondii. The pathogenicity of this species depends on the type originating from the clonal population. Techniques for more accurately determining the type of T. gondii have recently been developed using genetic markers. Specifically, T. gondii has been typed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). This study aimed to identify sets of PCR-RFLP markers that have high power to discriminate genotyping of T. gondii and are easy to use and are easy to use. The objective of this study was to characterize virulent strain isolates of T. gondii by PCR-RFLP using 10 markers with DdeI. Materials and Methods: T. gondii tachyzoites (RH virulent strain) were derived from culture cells at the Indonesian Research Center for Veterinary Sciences. Genotyping was performed on T. gondii DNA extracted from cell cultured tachyzoites using 10 genetic markers of PCR-RFLP, namely, B1#1, B1#2, B1#3, SAG1#1, SAG1#2, P30, BAG1, ROP1, GRA1, and GRA7, with digestion using the restriction enzyme DdeI. Results: The 10 genes were amplified by PCR. Among them, three genetic markers, B1#3, ROP1, and GRA1, were genotyped by the PCR-RFLP using restriction enzyme DdeI. Overall, the findings showed that the specific RFLP profile of digestion of gene regions by DdeI could be used as a specific marker for the virulent biotype causative of toxoplasmosis. In addition, virulent strains of T. gondii can be easily detected by these markers. Conclusion: Three pairs of primers (B1#3, ROP1, and GRA1) with DdeI have proven useful for the diagnosis of acute toxoplasmosis (virulent strain biotype I). This proposed method is relatively simple, rapid, cheap, and can be performed in most laboratories, providing a practical approach for the routine analysis of T. gondii strains.

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.

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