Computer-simulated virtual RFLP patterns generated from in silico digestions of phytoplasma 16S rRNA genes from strains detected in this study belonging to groups: (A) 16SrI using HhaI (MH428961: 16SrI-P *); (B) 16SrIII using BstUI (MH428959: 16SrIII-F *); (C) 16SrIX using RsaI
(MH428962:16SrIX-A[infinito]); (D) 16SrXIII using MseI (MH428958: 16SrXIII-H *); (E, F) 16SrXV using HaeIII and HpalI (MH428963: 16SrXV-A[infinito]), obtained using iPhyClassifier tool.
The anticipated patterns of the 3 enzymes include the cleavage of exon 2 and 3 with BbvI and RsaI
PCR-RFLP analysis: 6 uL of each PCR product of the rDNA-ITS was digested with one of the following 8 restriction enzymes: AluI, BsuRI, CfoI, EcoRI, HinfI, PstI, RsaI
and TaqI (Takara bioinc.) in the buffer stipulated by the manufacture.
(20), with the enzymes PstI (Promega, USA), HaeIII (Promega, Madison USA), DdeI (Promega), and RsaI
In this study, PCR-RFLP based on the partial rDNA of ITS1 and restriction RsaI
enzyme was used for differentiation and identification of Fasciola species in Nghe An province.
Paper presented at the 52nd European Congress of the RSAI
For RFLP analysis, the resultant 450 base pairs PCR products were digested by six restriction enzymes (BamHI, DdeI, HaeIII, HinfI, PstI, RsaI
; Invitrogen, Sao Paulo, Brazil).
We used RsaI
and a cleavage protocol following the manufacturer's instructions.
Genotyping of this variant was performed by amplification from 50 to 100 ng of genomic DNA, followed by digestion using RsaI
For patient 3, a total of 8 [micro]l of the long template PCR product (Supplementary Table 1) were double digested 1 hour at 37[degrees]C with 1X digestion buffer Tango (Fermentas, Burlington, Ontario, Canada) and 5 U of RsaI
(Fermenta) and PstI (New England Biolabs).
In this assay, two pairs of primers, one specific to 57:01 allele and another pair is for control, selectively amplify genomic DNA followed by digestion with restriction enzymes NlaIII or RsaI
. PCR products amplified from two different pairs of primers resulted in different sizes of fragments that can be visualized easily on the agarose gel.
DNA was digested using HinfI and RsaI
enzymes and the digested DNA separated on 0.8% agarose gels.