ÿþ<HTML><HEAD><TITLE>25º Congresso Brasileiro de Microbiologia </TITLE><link rel=STYLESHEET type=text/css href=css.css></HEAD><BODY aLink=#ff0000 bgColor=#FFFFFF leftMargin=0 link=#000000 text=#000000 topMargin=0 vLink=#000000 marginheight=0 marginwidth=0><table align=center width=700 cellpadding=0 cellspacing=0><tr><td align=left bgcolor=#cccccc valign=top width=550><font face=arial size=2><strong><font face=Verdana, Arial, Helvetica, sans-serif size=3><font size=1>25º Congresso Brasileiro de Microbiologia </font></font></strong><font face=Verdana size=1><b><br></b></font><font face=Verdana, Arial,Helvetica, sans-serif size=1><strong> </strong></font></font></td><td align=right bgcolor=#cccccc valign=top width=150><font face=arial size=2><strong><font face=Verdana, Arial, Helvetica, sans-serif size=1><font size=1>ResumoID:1762-1</font></em></font></strong></font></td></tr><tr><td colspan=2><br><br><table align=center width=700><tr><td>Área: <b>Microbiologia Geral ( Divisão H )</b><p align=justify><strong><FONT FACE="TIMES NEW ROMAN, TIMES, SERIF">MOLECULAR TYPING METHOD OF <EM>LEPTOSPIRA INTERROGANS</EM> SEROVARS</FONT></strong></p><p align=justify><b>Josefa Bezerra da Silva </b> (<i>Instituto Butantan</i>); <b>Eneas de Carvalho </b> (<i>Instituto Butantan</i>); <b>Zenaide Maria de Morais </b> (<i>USP</i>); <b><u>Regiane Degan Favaro </u></b> (<i>Instituto Butantan</i>); <b>Sílvio Arruda Vasconcellos </b> (<i>USP</i>); <b>Paulo Lee Ho </b> (<i>Instituto Butantan</i>)<br><br></p><b><font size=2>Resumo</font></b><p align=justify class=tres><font size=2><P class=MsoNormal style="MARGIN: 0cm 0cm 0pt; TEXT-ALIGN: justify"><SPAN lang=EN-US style="mso-ansi-language: EN-US">Leptospirosis is the most widespread zoonotic disease in the world. There are about 200 pathogenic serovars of <SPAN style="mso-bidi-font-style: italic"><EM>Leptospira</EM></SPAN>, the bacteria that cause leptospirosis. The serovar classification is based on antigenic determinants, mostly related to lipopolysaccharides (LPS). Nevertheless, other classification methods are currently being proposed. Since the difference in the LPS composition observed among <EM>Leptospira</EM> serovars can be a result of the differences present in their LPS biosynthesis loci, the amplification of these genes can be a powerful tool to associate the serological and the molecular classifications. In this work, we investigated if the amplification of the LPS biosynthesis loci can provide adequate information for Leptospira typing. The total genomic DNA was isolated from <SPAN style="mso-bidi-font-style: italic"><EM>L. interrogans</EM></SPAN> serovars Copenhageni, Canicola, Hardjo, Bataviae, Lai, Naam, Pyrogenes, Australis, Autumnalis, Smith and Mwogolo. Paired primers were designed based on the nucleotide sequence of the <EM>rfb</EM> loci from diverse leptospires serovars. The polymorphic regions were chosen for primer design, to promote differential amplifications among the serovars. An <EM>in silico</EM> analysis was used to confirm if the fragments amplified will, indeed, differ among some <EM>Leptospira </EM>serovars. The polymerase chain reaction (PCR) was carried out using single pairs primer or using multiplexed primers. <SPAN style="mso-bidi-font-weight: bold">The annealing cycle was performed in two conditions: 45º and 52º C.<SPAN style="mso-spacerun: yes">&nbsp; </SPAN></SPAN>Several nonspecific amplifications were observed in the two annealing conditions. Even so, the amplifications using four selected primer pairs (at <SPAN style="mso-bidi-font-weight: bold">45º C) were efficient to differentiate three serovars and also to differentiate three groups of serovars from all others. The serovars and groups of serovars that can be differentiated were: (1) </SPAN>Hardjo; (2) Australis; (3) Bataviae; (4) Copenhageni, Mwogolo, Smith and Lai; (5) Naam and Autumnalis; (6) Canicola and Pyrogenes. However, when these primers were multiplexed in a single PCR (using Copenhageni as the template DNA), a nonspecific fragment, that was not present in the reactions using single primers, was unexpected amplified. Thus the use of primers that can amplify the LPS biosynthesis loci is a method able to link the serological classification to a molecular one. Nevertheless, the amplification of nonspecific fragments was a difficulty not yet transposed step.</SPAN></P> <P class=MsoNormal style="MARGIN: 0cm 0cm 0pt; TEXT-ALIGN: justify"><SPAN lang=EN-US style="mso-ansi-language: EN-US"></SPAN>&nbsp;</P><SPAN lang=EN-US style="mso-ansi-language: EN-US"> <P class=MsoNormal style="MARGIN: 0cm 0cm 0pt; TEXT-ALIGN: justify"><SPAN lang=EN-US style="mso-ansi-language: EN-US">Supported by FAPESP, CNPq and Fundação Butantan<?xml:namespace prefix = o ns = "urn:schemas-microsoft-com:office:office" /><o:p></o:p></SPAN></P><o:p></o:p></SPAN> <P class=MsoNormal style="MARGIN: 0cm 0cm 0pt"><SPAN lang=EN-US style="mso-ansi-language: EN-US"><o:p>&nbsp;</o:p></SPAN></P> <P>&nbsp;</P></font></p><br><b>Palavras-chave: </b>&nbsp;Leptospirosis, Leptospira, polymerase chain reaction, molecular typing</td></tr></table></tr></td></table></body></html>