REDigest: a Python GUI for In-Silico Restriction Digestion Analysis of Genes or Complete Genome Sequences

Restriction fragment length polymorphism (RFLP) is a technology for the molecular characterization of DNA and widely used genome mapping, medical genetics, molecular microbiology and forensics etc. Terminal restriction fragment length polymorphism (T-RFLP), a variant of RFLP is extensively used in environmental microbiology for the microbial community profiling based on the restriction digestion profile of marker gene (16S rRNA, FTHFS etc.) amplicons. At present, there is a lack of a tool which can perform in-silico restriction digestion of a large number of sequences at a time, in an interactive way and as an output produce sequences of the restriction fragments and visualization plot. I have developed a graphical user interface based software REDigest for the in-silico restriction digestion analysis for gene or genome sequences. The REDigest software program with a graphical user interface is freely available at https://github.com/abhijeetsingh1704/REDigest.

Genotyping of Cryptosporidium species in children suffering from diarrhea in Sharkyia Governorate, Egypt

Introduction: The protozoan parasite Cryptosporidium is one of the principal reasons for childhood diarrhea around the world. This work aimed to differentiate Cryptosporidium species among children suffering from diarrhea in Sharkyia Governorate, Egypt. Methodology: A total of 97 fecal specimens were taken from children suffering from diarrhea, attending Pediatric Clinics of Zagazig University and Al-Ahrar Hospitals. Full history was taken. Stool samples were examined microscopically using modified Ziehl–Neelsen stain for detection of Cryptosporidium oocysts. To identify Cryptosporidium genotypes, positive samples were then subjected to nested Polymerase chain reaction-restriction fragment length polymorphism targeting Cryptosporidium oocyst wall protein gene. Results: The overall detection rate was 27.8% (27/97) using modified Ziehl–Neelsen stain staining method. Using nested polymerase chain reaction, the gene was amplified in 85.2% (23/27). Restriction fragment length polymorphism analysis revealed that 65.2% (15/23) were Cryptosporidium hominis, 30.4% (7/23) were Cryptosporidium parvum, and one sample was not typed (4.4%). The significant risk factors associated with Cryptosporidium infection in children were animal contact and residence in rural areas. Conclusions: Cryptosporidium is a common enteric parasite affecting children in Sharkyia Governorate, Egypt, with the predominance of C. hominis genotype in children.

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

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.

Comparative lactic acid bacteria (LAB) profiles during dadih fermentation with spontaneous and back-slopping methods, as identified by terminal-restriction fragment length polymorphism (T-RFLP)

The diversity of lactic acid bacteria (LAB) present during the manufacture of traditional fermented buffalo milk from West Sumatra, known as dadih, was studied via a culture-independent approach using terminal-restriction fragment length polymorphism (T-RFLP) to compare the dynamic diversity in back-slopping and spontaneous fermentation methods. Total LAB and pH were measured in freshly prepared buffalo milk and in \textit{dadih} fermented for 24 and 48 hours. The results indicated significant differences between the fermentation methods, with higher total LAB, and greater phylotype richness and relative abundance being identified in the back-slopping method. Terminal fragment lengths (TRFs) of 68 and 310 bp were common to both techniques, similar to those of Lactobacillus fermentum, Fructobacillus pseudoficulneus, Leuconostoc citreum, Leuconostoc kimchii, and Leuconostoc sp. The changes in phylotype number (species number) and relative abundances of LAB communities identified are expected to produce data needed to formulate the best fermentation process for dadih manufacturing. A 24-hour back-slopping fermentation method is recommended, as fermentation time of longer than 24 hours reduced viable LAB significantly. Our results also indicated that the T-RFLP technique is not only clearly sensitive enough and adequate for segregating LAB diversity in both fermentation methods, but that it also provides good information regarding the structure of microbial communities and their composition change during the fermentation process.

Application of polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR) in the analysis of single nucleotide polymorphisms (SNPs)

Polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR) is a technique used to identify single nucleotide polymorphisms (SNPs) based on the recognition of restriction sites by restriction enzymes. RFLP-PCR is an easy-to-perform and inexpensive tool for initial analysis of SNPs potentially associated with some monogenic diseases, as well as in genotyping, genetic mapping, lineage screening, forensics and ancient DNA analysis. The RFLP-PCR method employs four steps: (1) isolation of genetic material and PCR; (2) restriction digestion of amplicons; (3) electrophoresis of digested fragments; and (4) visualisation. Despite its obsolescence and the presence of high-throughput DNA analysis techniques, it is still applied in the analysis of SNPs associated with disease entities and in the analysis of genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RFLP-PCR is a low-cost and low-throughput research method allowing for the analysis of SNPs in the absence of specialised equipment, and it is useful when there is a limited budget.

Evaluation of PCR pncA-restriction fragment length polymorphism and PCR amplification of genomic regions of difference for the identification of M. bovis strains in lymph nodes cultures

Background: A rapid accurate identification of Mycobacterium bovis is essential for surveillance purposes. Objectives: A PCR pncA-Restriction Fragment Length Polymorphism (RFLP) and a multiplex PCR based on the detection of 3 regions of difference (RD-PCR): RD9, RD4 and RD1 were evaluated for the identification of M. bovis in lymph nodes cultures, in Tunisia, during 2013-2015. Methods: Eighty-two M. tuberculosis complex strains were identified using the biochemical tests, GenoType MTBC assay, PCR pncA-RFLP and RD-PCR. Results: The PCR pncA-RFLP showed that 54 M. bovis strains, identified by GenoType MTBC, had a mutation at position 169 of pncA gene. Twenty-eight strains did not show any mutation at this position 27 M. tuberculosis isolates and one M. caprae. The PCR pncA-RFLP had a sensitivity of 100.0% (95%CI: 93.3 -100.0) and a specificity of 100.0% (95%CI: 87.9- 100.0) for identifying M. bovis. The RD-PCR showed that all M. bovis strains had the RD9 and RD4 deleted but presented RD1. RD-PCR also presented high sensitivity and specificity in detecting M. bovis strains (100.0%). Conclusions: PCR pncA-RFLP and RD-PCR represent very accurate and rapid tools to identify M. bovis. They can be easily implemented in each laboratory due to their low cost and easy use. Keywords: GenoType MTBC; lymph nodes; Mycobacterium bovis; PCR pncA-RFLP; RD-PCR.

Identification of Zoonotic Balantioides coli in Pigs by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Its Distribution in Korea

Balantioides coli is a zoonotic protozoan parasite whose main reservoir is pigs. Recent studies have shown that B. coli variant A but not B has zoonotic potential. While B. coli infection has been reported in different animals and countries, the prevalence of the zoonotic variant is limited due to a lack of molecular information. Therefore, this study investigated the prevalence of B. coli in domestic pigs in Korea and assessed its zoonotic potential. A total of 188 pig fecal samples were collected from slaughterhouses in Korea. B. coli was identified by microscopy and molecular methods. B. coli was identified in 79 (42.9%) and 174 (94.6%) samples by microscopy and polymerase chain reaction (PCR), respectively. This study also developed a PCR-restriction fragment length polymorphism (PCR-RFLP) method to differentiate B. coli variant A from B without sequence analysis. Using this method, 62 (33.7%) and 160 (87.0%) samples were positive for variants A and B, respectively, and 48 (26.1%) samples were co-infected with both variants. Sequence and phylogenetic analyses showed a high genetic diversity of B. coli in pigs in Korea. To our knowledge, this is the first study to develop a method to differentiate B. coli variants A and B without sequence analysis and to assess the molecular epidemiology of B. coli in pigs. Continuous monitoring of zoonotic B. coli in pigs should be performed as pigs are the main source of human balantidiasis.

Detection of Cryptosporidium in stool sample by PCR-RFLP v1

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.

Comparison of Whole Genome Sequencing versus Standard Molecular Diagnostics for Species Identification in the Leishmania Viannia Subgenus

The prognosis and treatment of New World tegumentary leishmaniasis is dependent on the infecting species, yet such species identification in the Leishmania Viannia subgenus poses a diagnostic challenge. Currently, speciation relies on standard molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, and Sanger sequencing (SS). Whole-genome sequencing (WGS) is a robust and increasingly cost-efficient tool that may improve Leishmania species identification. We evaluated WGS versus standard RFLP-SS for species identification in three reference and five clinical strains of Leishmania Viannia spp. Internal transcribed spacer1 (its1), cysteine proteinase b (cpb), and heat shock protein 70 (hsp70) polymerase chain reaction-restriction fragment length polymorphism (RFLP) was performed, followed by SS of the its2, cpb, hsp70, and mannose phosphate isomerase (mpi) loci. After de novo assembly, sequences were mapped, and homology compared with both reference strains and reference genomes on National Center for Biotechnology Information. All American Type Culture Collection strains were confirmed to be single-species of L. V. braziliensis, L. V. guyanensis, or L. V. panamensis by WGS. Conversely, RFLP-SS was able to definitively identify one of three isolates to the species level. Clinical samples were identified as either single-species (N = 3), mixed (N = 1), or hybrid (N = 1) infections by WGS, while standard molecular diagnosis required multi-target composite analysis for identification due to loci-dependent results by RFLP-SS. We have corroborated the utility of WGS as a diagnostic tool to speciate members of the L. Viannia subgenus and to discriminate between mixed and hybrid infections. WGS is a potentially useful complement to multistaged RFLP-SS for species identification in Leishmania infections.