Study on the Detection of rs9263726 Locus by Nested PCR-RFLP

In July 2015, 179 grapevine plants belonging to 16 grapevine autochthonous cultivars were assessed for sanitary status using DAS ELISA test for the presence of: Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2)and Grapevine leafroll-associated virus 3 (GLRaV-3). Furthermore, surveyfor the phytoplasma presence and laboratory analyses using nested-PCR/RFLP assay was conducted at the beginning of September 2015 on grapevine cultivars which were not positive in DAS ELISA test for the presence of the four viruses. Out of 179 tested plants with DAS ELISA test, 146 (81%) were positive for the presence of at least one virus. The most widespread viruses were GFLaV- 1 and GFLaV- 3 with approximately 80 % of grapevines infected. Nested–PCR/RFLP assay showed that out of 33 tested samples 2 were positive for the presence of phytoplasmas from 16SrXII group. Sanitation of infected grapevine cultivars is needed in near future.

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Assessment of Giardia and Cryptosporidium Assemblages/ Species and Their Viability in Potable Tap Water in Beni-Suef, Egypt Using Nested PCR/RFLP and Staining

Iranian Journal of Parasitology ◽

10.18502/ijpa.v14i3.1475 ◽

2019 ◽

Author(s):

Doaa HAMDY ◽

Ayman El-BADRY ◽

Wegdan ABD EL WAHAB

Keyword(s):

Water Samples ◽

Rural Areas ◽

Nested Pcr ◽

Membrane Filtration ◽

Tap Water ◽

Significant Risk ◽

Waterborne Diseases ◽

Filtration Method ◽

Pcr Rflp ◽

Blue Stain

Background: The protozoan Giardia and Cryptosporidium are responsible for most water-borne diseases all over the world. The extent and number of outbreaks of waterborne diseases suggests a significant risk of their potential transmission via drinking water. This study aimed to document the prevalence and viability of Giardia and Cryptosporidium (oo) cysts in tap water samples in Beni-Suef Governorate, Egypt and to detect the predominant Giardia and Cryptosporidium assemblages/species using nested PCR/ Restriction Fragment Length Polymorphism (RFLP) confirmed by further sequencing of positive samples. Methods: A total of 80 tap water samples were collected throughout a year from four big centers and filtered using the membrane filtration method. Samples were stained by Lugol’s iodine, Modified Zeihl-Neelsen (MZN) (to detect prevalence) and trypan blue stain (to detect viability). Nested PCR-RFLP and sequencing were used for molecular characterizations and genotyping of the detected Giardia and Cryptosporidium. Results: Giardia and Cryptosporidium DNA was detected in 20 (25%) and 29 (36.3%) samples respectively, with predominance of Giardia assemblage B (85%) and C. hominis (75.9%). The prevalence and viability of both parasites (oo) cysts showed seasonality which peaked in summer and were greater in Beba center and in rural areas. Conclusion: To our knowledge, no studies have been done in these areas before. The anthroponotic transmission has an important role in giardiasis and cryptosporidiosis epidemiology in this studied area.

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Molecular Characterization ofCryptosporidiumspp. in Wild Rodents of Southwestern Iran Using 18s rRNA Gene Nested-PCR-RFLP and Sequencing Techniques

Journal of Tropical Medicine ◽

10.1155/2016/6834206 ◽

2016 ◽

Vol 2016 ◽

pp. 1-6 ◽

Cited By ~ 12

Author(s):

Jasem Saki ◽

Masoud Foroutan-Rad ◽

Reza Asadpouri

Keyword(s):

Molecular Characterization ◽

Nested Pcr ◽

18S Rrna ◽

Restriction Enzymes ◽

18S Rrna Gene ◽

Rrna Gene ◽

Wild Rodents ◽

Nested Polymerase Chain Reaction ◽

Pcr Rflp ◽

Southwest Of Iran

Background. Rodents could act as reservoir forCryptosporidiumspp. speciallyC. parvum, a zoonotic agent responsible for human infections. Since there is no information aboutCryptosporidiuminfection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization ofCryptosporidiumspp. in this region.Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method usingSspIandVspIrestriction enzymes was carried out to genotype the species and then obtained results were sequenced.Results. Three out of 100 samples were diagnosed as positive and overall prevalence ofCryptosporidiumspp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples andC. parvumpattern was identified. Finally PCR-RFLP findings were sequenced and presence ofC. parvumwas confirmed again.Conclusions. Our study showed rodents could be potential reservoir forC. parvum. So an integrated program for control and combat with them should be adopted and continued.

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