<?xml version="1.0" encoding="ISO-8859-1"?><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" xmlns:xsi="http://www.w3.org/2001/XMLSchema-instance">
<front>
<journal-meta>
<journal-id>1665-1456</journal-id>
<journal-title><![CDATA[Biotecnia]]></journal-title>
<abbrev-journal-title><![CDATA[Biotecnia]]></abbrev-journal-title>
<issn>1665-1456</issn>
<publisher>
<publisher-name><![CDATA[Universidad de Sonora, División de Ciencias Biológicas y de la Salud]]></publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id>S1665-14562025000100138</article-id>
<article-id pub-id-type="doi">10.18633/biotecnia.v27.2495</article-id>
<title-group>
<article-title xml:lang="en"><![CDATA[The Not-alike3 command pipeline finds genomic targets for the molecular diagnosis of diseases caused by pathogen microorganisms]]></article-title>
<article-title xml:lang="es"><![CDATA[La tubería de comandos Not-alike3 encuentra blancos genómicos para el diagnóstico molecular de enfermedades causadas por microorganismos patógenos]]></article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Montalvo-Arredondo]]></surname>
<given-names><![CDATA[Javier]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Juárez-Verdayes]]></surname>
<given-names><![CDATA[Marco A.]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname><![CDATA[Rivas-Martínez]]></surname>
<given-names><![CDATA[Erika Nohemi]]></given-names>
</name>
<xref ref-type="aff" rid="Aff"/>
</contrib>
</contrib-group>
<aff id="Af1">
<institution><![CDATA[,Universidad Autónoma Agraria Antonio Narro Molecular Bioengineering and Bioinformatics Laboratory Departamento de Ciencias Básicas]]></institution>
<addr-line><![CDATA[Saltillo Coahuila]]></addr-line>
<country>Mexico</country>
</aff>
<aff id="Af2">
<institution><![CDATA[,Universidad Autónoma Agraria Antonio Narro Departamento de Botánica ]]></institution>
<addr-line><![CDATA[Saltillo Coahuila]]></addr-line>
<country>Mexico</country>
</aff>
<pub-date pub-type="pub">
<day>00</day>
<month>12</month>
<year>2025</year>
</pub-date>
<pub-date pub-type="epub">
<day>00</day>
<month>12</month>
<year>2025</year>
</pub-date>
<volume>27</volume>
<copyright-statement/>
<copyright-year/>
<self-uri xlink:href="http://www.scielo.org.mx/scielo.php?script=sci_arttext&amp;pid=S1665-14562025000100138&amp;lng=en&amp;nrm=iso"></self-uri><self-uri xlink:href="http://www.scielo.org.mx/scielo.php?script=sci_abstract&amp;pid=S1665-14562025000100138&amp;lng=en&amp;nrm=iso"></self-uri><self-uri xlink:href="http://www.scielo.org.mx/scielo.php?script=sci_pdf&amp;pid=S1665-14562025000100138&amp;lng=en&amp;nrm=iso"></self-uri><abstract abstract-type="short" xml:lang="en"><p><![CDATA[Abstract DNA-based molecular techniques are crucial for the precise identification of microorganisms. Despite their speed and sensitivity, specificity hinges on primer or probe design. The search for unique sequences in organisms&#8217; genomes has various applications such as microorganism identification. There are published bioinformatic tools that employ the concept of in silico genomic subtractive hybridization analysis to identify species-specific/unique regions within a genome of interest. However, they use deprecated tools and programming languages that are not currently used in bioinformatics, some of them are specialized or difficult to obtain because the repositories, where these tools were hosted, are not public or the web servers are currently down. To address these issues, we implemented the in silico genomic subtractive hybridization idea in a user-friendly, open-source, freely available, easy-to-install command pipeline application written in Python (called Not-alike3). We designed PCR primers over the sequence of unique regions identified in the genomes of two Mucorales species, Rhizopus oryzae and Cunninghamella bertholletia, employing Not-alike3 command pipeline; we made specificity tests to challenge these primers and we observed that they were species-specific.]]></p></abstract>
<abstract abstract-type="short" xml:lang="es"><p><![CDATA[Resumen Las técnicas moleculares basadas en ADN son cruciales para la identificación correcta de microorganismos. A pesar de su velocidad y sensibilidad, la especificidad depende en el diseño de la sonda o el primer de PCR. La búsqueda por secuencias únicas en gnomas de organismos tiene varias aplicaciones como la misma identificación de microorganismos. Existen herramientas bioinformáticas publicadas que emplean el concepto de hibridación genómica sustractiva in silico para la identificación de regiones únicas o específicas de especie dentro de un genoma de interés. Sin embargo, éstas usan herramientas desactualizadas y/o lenguajes de programación que actualmente no son usados en el área de la bioinformática, otras herramientas son muy especializadas o son difíciles de obtener debido a que los repositorios (acervos) donde se hospedan las herramientas no son públicas o están apagados actualmente. Para abordar estos problemas, implementamos el concepto de hibridación genómica sustractiva in silico en una tubería de comandos escrita en Python que es amigable, de código abierto, gratis y fácil de instalar (llamada Not-alike3). Adicionalmente, diseñamos primers de PCR utilizando como molde la secuencia de regiones únicas identificadas en los genomas de dos especies de Mucorales, Rhizopus oryzae y Cunninghamella bertholletiae empleando la tubería Not-alike3, después realizamos evaluaciones de especificidad para desafiar esos primers que diseñamos y observamos que dichos primers fueron específicos de especie.]]></p></abstract>
<kwd-group>
<kwd lng="en"><![CDATA[Python programming]]></kwd>
<kwd lng="en"><![CDATA[molecular diagnostics]]></kwd>
<kwd lng="en"><![CDATA[pathogen detection]]></kwd>
<kwd lng="en"><![CDATA[molecular biology]]></kwd>
<kwd lng="en"><![CDATA[SHG]]></kwd>
<kwd lng="es"><![CDATA[programación Python]]></kwd>
<kwd lng="es"><![CDATA[diagnóstico molecular]]></kwd>
<kwd lng="es"><![CDATA[detección de patógenos]]></kwd>
<kwd lng="es"><![CDATA[biología molecular]]></kwd>
<kwd lng="es"><![CDATA[SHG]]></kwd>
</kwd-group>
</article-meta>
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