Molecular Profile of Trichophyton mentagrophytes and Microsporum canis based on PCR-RFLP of Internal Transcribed Spacer

Abstract

<p><em>Trichophyton mentagrophytes</em> and <em>Microsporum canis</em> are dermatophytes fungi which commonly infect animal and human. Conventional and molecular methods were used for identification of the fungus. The region of internal transcribed spacer (ITS) has a high probability for fungal identification. PCR-RFLP was reported as a useful method to differentiate dermatophytes fungi. The objective of the study was<em> </em>to compare molecular profile of <em>T. mentagrophytes</em> and <em>M. canis</em> based on the result of ITS fragment digestion using Dde I, Hinf I and Mva I. The molds were isolated from skin scrapping of 18 animals which showed dermatophytosis lesion. The isolated molds were grown on agar plate for 14 days of incubation at 37<sup>o</sup>C and then identified based on macro and microscopic morphologies. Amplification of chitin synthase gene was used for confirmation and separation of dermatophytes from other fungi. ITS fragment was amplified and then digested using restriction enzymes Dde I, Hinf I and Mva I. The result showed that digestion products from ITS fragment of <em>T. mentagrophytes</em> and <em>M. canis </em>were different<em>.</em> The fragment 159 bp from Dde I, 374 bp from Hinf I and 89 bp from Mva I were present in <em>T. mentagrophytes</em> but absent in <em>M. canis</em>. Based on these results, specific RFLP profile of digestion ITS region by Dde I, Hinf I and Mva I can be used as a specific marker for species of dermatophytes fungi.</p>

The Patterns of Restriction Fragment of Several Enzymes to Distinguish Toxoplasma gondii Isolates Virulent and Avirulent Strains using GRA1 and GRA7 Genetic Marker

“…Generally, enzymes that can be used by both genes simultaneously to differentiate genotypes namely MvaI and HinfI enzymes can be used to digest GRA1 and GRA7 genes to differentiate T. gondii genotypes I and II. The sequence influences the location of the cleavage site, resulting in a variation in the size of the digested products (Endrawati et al 2021). The examination of the restriction fragment length distribution is conducted in silico, or on a computer.…”

“…In silico analysis typically forecasts how they will appear in the experiment. Because the T. gondii genome sequence is publicly available, these methods may be quickly described and optimized using in silico modeling (Endrawati et al 2021;Ekawasti et al 2021Ekawasti et al , 2022. Due to insufficient digestion, in silico prediction analysis and electrophoresis produced different conclusions about the restriction sites.…”

“…The concentration of the missing fragments may be quite low, and the electrophoresis gel did not show any signs of them. Moreover, it continued to leave uncut fragments in enzyme digestion that may have been brought on by the disease or an inefficient digestion process (Endrawati et al 2021). As the concentration of the PCR result was not assessed, low-molecularweight fragments, the optimization approach (the circumstances or digestion time being not optimal), and the ratio of enzyme and DNA may not be suitable (Endrawati et al 2021;Ekawasti et al 2021).…”

“…Moreover, it continued to leave uncut fragments in enzyme digestion that may have been brought on by the disease or an inefficient digestion process (Endrawati et al 2021). As the concentration of the PCR result was not assessed, low-molecularweight fragments, the optimization approach (the circumstances or digestion time being not optimal), and the ratio of enzyme and DNA may not be suitable (Endrawati et al 2021;Ekawasti et al 2021). a very low concentration that makes the electrophoresis gel undetectable (Endrawati et al 2021).…”

Soursop leaves (Annona muricata L.) endophytic fungi anticancer activity against HeLa cells

“…A polymerase chain reaction (PCR) was then conducted to amplify the ITS region of the extracted DNA. Two ITS primers were used, i.e., ITS Primer 4 (reverse): 5′–TCC TCC GCT TAT TGA TAT GC–3′, and ITS Primer 5 (forward): 5′–GGA AGT AAA AGT CGT AAC AAG G–3′ ( Endrawati and Kusumaningtyas, 2021 , Ausubel et al, 2004 , McBreen et al, 2003 ). Amplification was conducted on 10 µL consisting of dNTP of 0.2 mM, DreamTaq 1 × buffer (containing MgCl 2 ), ITS4 Primer of 0.5 pmol, ITS5 Primer of 0.5 pmol, Taq polymerase of 0.125 U, DNA template of 100 ng, and NFW of 5.75 µL.…”