Comparison of heat shock protein 70 kDa and 18S rDNA genes for molecular detection and phylogenetic analysis of Babesia vogeli from whole blood of naturally infected dogs
A total of 300 blood samples of domiciliated dogs in rural and urban areas of southeast Rio de Janeiro State, Brazil, were used to compare the 18S ribosomal DNA region (18S rDNA) and the heat shock protein 70 kDa (hsp70) gene for molecular detection of Babesia vogeli and to perform a phylogenetic st...
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| creator | Paulino, Patrícia G. Pires, Marcus S. Silva, Claudia B. da Peckle, Maristela Costa, Renata L. da Vitari, Gabriela L.V. Abreu, Ana Paula M. de Almosny, Nádia R.P. Massard, Carlos L. Santos, Huarrisson A. |
| description | A total of 300 blood samples of domiciliated dogs in rural and urban areas of southeast Rio de Janeiro State, Brazil, were used to compare the 18S ribosomal DNA region (18S rDNA) and the heat shock protein 70 kDa (hsp70) gene for molecular detection of Babesia vogeli and to perform a phylogenetic study comparing the two genes for B. vogeli classification. Using conventional polymerase chain reaction (cPCR) of 18S rDNA and hsp70 sequences, we were able to detect B. vogeli with the same sensitivity (96.15%) and specificity (99.63%). However, sequencing revealed one false positive (Rangelia sp.) for 18S rDNA that was not detected by hsp70. This is the first report of an organism closely related to the Rangelia vitalii parasite of dogs in Brazil. In the hsp70-cPCR and hsp70-qPCR comparison, 15.66% of samples were considered positive by quantitative (q)PCR, significantly more than was detected by cPCR (8.66%). In addition to the high conservation of the 18S rDNA, phylogenetic analysis showed that the hsp70 gene can be used to describe phylogenetic relationships between canine piroplasmids with more accuracy than 18S rDNA. According to these findings, the qPCR method has greater sensitivity than cPCR for detection of B. vogeli in naturally infected dogs. The hsp70-qPCR detection limit was 10 copies, with an efficiency of 100.30% and a determination coefficient (R2) of 0.998. The development of this qPCR method provides a highly sensitive approach for B. vogeli molecular detection and a tool that is capable of quantifying parasitemia levels in whole blood samples from dogs. The primers and probes were designed to be specific for B. vogeli, though analytical specificity of the assay has not been tested in vitro with DNA of certain Babesia species that infect dogs. The hsp70 gene is a precise molecular marker for Babesia phylogeny, especially species that infect dogs. |
| doi_str_mv | 10.1016/j.ttbdis.2018.01.013 |
| format | Article |
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Using conventional polymerase chain reaction (cPCR) of 18S rDNA and hsp70 sequences, we were able to detect B. vogeli with the same sensitivity (96.15%) and specificity (99.63%). However, sequencing revealed one false positive (Rangelia sp.) for 18S rDNA that was not detected by hsp70. This is the first report of an organism closely related to the Rangelia vitalii parasite of dogs in Brazil. In the hsp70-cPCR and hsp70-qPCR comparison, 15.66% of samples were considered positive by quantitative (q)PCR, significantly more than was detected by cPCR (8.66%). In addition to the high conservation of the 18S rDNA, phylogenetic analysis showed that the hsp70 gene can be used to describe phylogenetic relationships between canine piroplasmids with more accuracy than 18S rDNA. According to these findings, the qPCR method has greater sensitivity than cPCR for detection of B. vogeli in naturally infected dogs. The hsp70-qPCR detection limit was 10 copies, with an efficiency of 100.30% and a determination coefficient (R2) of 0.998. The development of this qPCR method provides a highly sensitive approach for B. vogeli molecular detection and a tool that is capable of quantifying parasitemia levels in whole blood samples from dogs. The primers and probes were designed to be specific for B. vogeli, though analytical specificity of the assay has not been tested in vitro with DNA of certain Babesia species that infect dogs. The hsp70 gene is a precise molecular marker for Babesia phylogeny, especially species that infect dogs.</description><identifier>ISSN: 1877-959X</identifier><identifier>EISSN: 1877-9603</identifier><identifier>DOI: 10.1016/j.ttbdis.2018.01.013</identifier><identifier>PMID: 29409719</identifier><language>eng</language><publisher>Netherlands: Elsevier GmbH</publisher><subject>Animals ; Babesia - chemistry ; Babesia - genetics ; Babesia - isolation & purification ; Babesiosis ; Babesiosis - blood ; Babesiosis - diagnosis ; Babesiosis - epidemiology ; Babesiosis - parasitology ; Brazil - epidemiology ; DNA Primers - genetics ; DNA, Protozoan - genetics ; Dog Diseases - diagnosis ; Dog Diseases - epidemiology ; Dog Diseases - parasitology ; Dogs ; Heat shock protein 70 kDa ; HSP70 Heat-Shock Proteins - genetics ; Molecular Diagnostic Techniques - methods ; Phylogeny ; Real-time polymerase chain reaction ; Real-Time Polymerase Chain Reaction - methods ; RNA, Ribosomal, 18S - genetics</subject><ispartof>Ticks and tick-borne diseases, 2018-03, Vol.9 (3), p.556-562</ispartof><rights>2018 Elsevier GmbH</rights><rights>Copyright © 2018 Elsevier GmbH. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c362t-797634b7f5b8a748c44b88f59976b1f613b9ba051f8c0e0f9e0c5fafb9b723173</citedby><cites>FETCH-LOGICAL-c362t-797634b7f5b8a748c44b88f59976b1f613b9ba051f8c0e0f9e0c5fafb9b723173</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S1877959X17303655$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65534</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29409719$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Paulino, Patrícia G.</creatorcontrib><creatorcontrib>Pires, Marcus S.</creatorcontrib><creatorcontrib>Silva, Claudia B. da</creatorcontrib><creatorcontrib>Peckle, Maristela</creatorcontrib><creatorcontrib>Costa, Renata L. da</creatorcontrib><creatorcontrib>Vitari, Gabriela L.V.</creatorcontrib><creatorcontrib>Abreu, Ana Paula M. de</creatorcontrib><creatorcontrib>Almosny, Nádia R.P.</creatorcontrib><creatorcontrib>Massard, Carlos L.</creatorcontrib><creatorcontrib>Santos, Huarrisson A.</creatorcontrib><title>Comparison of heat shock protein 70 kDa and 18S rDNA genes for molecular detection and phylogenetic analysis of Babesia vogeli from whole blood of naturally infected dogs</title><title>Ticks and tick-borne diseases</title><addtitle>Ticks Tick Borne Dis</addtitle><description>A total of 300 blood samples of domiciliated dogs in rural and urban areas of southeast Rio de Janeiro State, Brazil, were used to compare the 18S ribosomal DNA region (18S rDNA) and the heat shock protein 70 kDa (hsp70) gene for molecular detection of Babesia vogeli and to perform a phylogenetic study comparing the two genes for B. vogeli classification. Using conventional polymerase chain reaction (cPCR) of 18S rDNA and hsp70 sequences, we were able to detect B. vogeli with the same sensitivity (96.15%) and specificity (99.63%). However, sequencing revealed one false positive (Rangelia sp.) for 18S rDNA that was not detected by hsp70. This is the first report of an organism closely related to the Rangelia vitalii parasite of dogs in Brazil. In the hsp70-cPCR and hsp70-qPCR comparison, 15.66% of samples were considered positive by quantitative (q)PCR, significantly more than was detected by cPCR (8.66%). In addition to the high conservation of the 18S rDNA, phylogenetic analysis showed that the hsp70 gene can be used to describe phylogenetic relationships between canine piroplasmids with more accuracy than 18S rDNA. According to these findings, the qPCR method has greater sensitivity than cPCR for detection of B. vogeli in naturally infected dogs. The hsp70-qPCR detection limit was 10 copies, with an efficiency of 100.30% and a determination coefficient (R2) of 0.998. The development of this qPCR method provides a highly sensitive approach for B. vogeli molecular detection and a tool that is capable of quantifying parasitemia levels in whole blood samples from dogs. The primers and probes were designed to be specific for B. vogeli, though analytical specificity of the assay has not been tested in vitro with DNA of certain Babesia species that infect dogs. The hsp70 gene is a precise molecular marker for Babesia phylogeny, especially species that infect dogs.</description><subject>Animals</subject><subject>Babesia - chemistry</subject><subject>Babesia - genetics</subject><subject>Babesia - isolation & purification</subject><subject>Babesiosis</subject><subject>Babesiosis - blood</subject><subject>Babesiosis - diagnosis</subject><subject>Babesiosis - epidemiology</subject><subject>Babesiosis - parasitology</subject><subject>Brazil - epidemiology</subject><subject>DNA Primers - genetics</subject><subject>DNA, Protozoan - genetics</subject><subject>Dog Diseases - diagnosis</subject><subject>Dog Diseases - epidemiology</subject><subject>Dog Diseases - parasitology</subject><subject>Dogs</subject><subject>Heat shock protein 70 kDa</subject><subject>HSP70 Heat-Shock Proteins - genetics</subject><subject>Molecular Diagnostic Techniques - methods</subject><subject>Phylogeny</subject><subject>Real-time polymerase chain reaction</subject><subject>Real-Time Polymerase Chain Reaction - methods</subject><subject>RNA, Ribosomal, 18S - genetics</subject><issn>1877-959X</issn><issn>1877-9603</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc-OFCEQxonRuJt138AYjl5mhKa7gYvJOrv-STZ6UBNvBOhih1m6GYFeMzcfyMR38kmkM7seJZVQKX71VagPoeeUrCmh_avduhQz-LxuCBVrQmuwR-iUCs5Xsifs8UPeyW8n6DznHamH0Vbw5ik6aWRLJKfyFP3axHGvk89xwtHhLeiC8zbaW7xPsYCfMCd_fv6-vdRYTwOm4jNOlx8v8A1MkLGLCY8xgJ2DTniAArb4qrSg--0hxAUr3taCDofs8zLjjTaQvcZ39TV47FIc8Y9tVcEmxDgsyKTLnHQIB-wnVzVhwEO8yc_QE6dDhvP7-wx9fXv1ZfN-df3p3YfNxfXKsr4pKy55z1rDXWeE5q2wbWuEcJ2sdUNdT5mRRpOOOmEJECeB2M5pV6u8YZSzM_TyqFt38H2GXNTos4UQ9ARxzopKKZu6T8Eq2h5Rm2LOCZzaJz_qdFCUqMUqtVNHq9RilSK0xtL24n7CbEYY_jU9GFOB10cA6j_vPCSVrYfJwuBTXYgaov__hL88U6oJ</recordid><startdate>201803</startdate><enddate>201803</enddate><creator>Paulino, Patrícia G.</creator><creator>Pires, Marcus S.</creator><creator>Silva, Claudia B. da</creator><creator>Peckle, Maristela</creator><creator>Costa, Renata L. da</creator><creator>Vitari, Gabriela L.V.</creator><creator>Abreu, Ana Paula M. de</creator><creator>Almosny, Nádia R.P.</creator><creator>Massard, Carlos L.</creator><creator>Santos, Huarrisson A.</creator><general>Elsevier GmbH</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>201803</creationdate><title>Comparison of heat shock protein 70 kDa and 18S rDNA genes for molecular detection and phylogenetic analysis of Babesia vogeli from whole blood of naturally infected dogs</title><author>Paulino, Patrícia G. ; Pires, Marcus S. ; Silva, Claudia B. da ; Peckle, Maristela ; Costa, Renata L. da ; Vitari, Gabriela L.V. ; Abreu, Ana Paula M. de ; Almosny, Nádia R.P. ; Massard, Carlos L. ; Santos, Huarrisson A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c362t-797634b7f5b8a748c44b88f59976b1f613b9ba051f8c0e0f9e0c5fafb9b723173</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Animals</topic><topic>Babesia - chemistry</topic><topic>Babesia - genetics</topic><topic>Babesia - isolation & purification</topic><topic>Babesiosis</topic><topic>Babesiosis - blood</topic><topic>Babesiosis - diagnosis</topic><topic>Babesiosis - epidemiology</topic><topic>Babesiosis - parasitology</topic><topic>Brazil - epidemiology</topic><topic>DNA Primers - genetics</topic><topic>DNA, Protozoan - genetics</topic><topic>Dog Diseases - diagnosis</topic><topic>Dog Diseases - epidemiology</topic><topic>Dog Diseases - parasitology</topic><topic>Dogs</topic><topic>Heat shock protein 70 kDa</topic><topic>HSP70 Heat-Shock Proteins - genetics</topic><topic>Molecular Diagnostic Techniques - methods</topic><topic>Phylogeny</topic><topic>Real-time polymerase chain reaction</topic><topic>Real-Time Polymerase Chain Reaction - methods</topic><topic>RNA, Ribosomal, 18S - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Paulino, Patrícia G.</creatorcontrib><creatorcontrib>Pires, Marcus S.</creatorcontrib><creatorcontrib>Silva, Claudia B. da</creatorcontrib><creatorcontrib>Peckle, Maristela</creatorcontrib><creatorcontrib>Costa, Renata L. da</creatorcontrib><creatorcontrib>Vitari, Gabriela L.V.</creatorcontrib><creatorcontrib>Abreu, Ana Paula M. de</creatorcontrib><creatorcontrib>Almosny, Nádia R.P.</creatorcontrib><creatorcontrib>Massard, Carlos L.</creatorcontrib><creatorcontrib>Santos, Huarrisson A.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Ticks and tick-borne diseases</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Paulino, Patrícia G.</au><au>Pires, Marcus S.</au><au>Silva, Claudia B. da</au><au>Peckle, Maristela</au><au>Costa, Renata L. da</au><au>Vitari, Gabriela L.V.</au><au>Abreu, Ana Paula M. de</au><au>Almosny, Nádia R.P.</au><au>Massard, Carlos L.</au><au>Santos, Huarrisson A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Comparison of heat shock protein 70 kDa and 18S rDNA genes for molecular detection and phylogenetic analysis of Babesia vogeli from whole blood of naturally infected dogs</atitle><jtitle>Ticks and tick-borne diseases</jtitle><addtitle>Ticks Tick Borne Dis</addtitle><date>2018-03</date><risdate>2018</risdate><volume>9</volume><issue>3</issue><spage>556</spage><epage>562</epage><pages>556-562</pages><issn>1877-959X</issn><eissn>1877-9603</eissn><abstract>A total of 300 blood samples of domiciliated dogs in rural and urban areas of southeast Rio de Janeiro State, Brazil, were used to compare the 18S ribosomal DNA region (18S rDNA) and the heat shock protein 70 kDa (hsp70) gene for molecular detection of Babesia vogeli and to perform a phylogenetic study comparing the two genes for B. vogeli classification. Using conventional polymerase chain reaction (cPCR) of 18S rDNA and hsp70 sequences, we were able to detect B. vogeli with the same sensitivity (96.15%) and specificity (99.63%). However, sequencing revealed one false positive (Rangelia sp.) for 18S rDNA that was not detected by hsp70. This is the first report of an organism closely related to the Rangelia vitalii parasite of dogs in Brazil. In the hsp70-cPCR and hsp70-qPCR comparison, 15.66% of samples were considered positive by quantitative (q)PCR, significantly more than was detected by cPCR (8.66%). In addition to the high conservation of the 18S rDNA, phylogenetic analysis showed that the hsp70 gene can be used to describe phylogenetic relationships between canine piroplasmids with more accuracy than 18S rDNA. According to these findings, the qPCR method has greater sensitivity than cPCR for detection of B. vogeli in naturally infected dogs. The hsp70-qPCR detection limit was 10 copies, with an efficiency of 100.30% and a determination coefficient (R2) of 0.998. The development of this qPCR method provides a highly sensitive approach for B. vogeli molecular detection and a tool that is capable of quantifying parasitemia levels in whole blood samples from dogs. The primers and probes were designed to be specific for B. vogeli, though analytical specificity of the assay has not been tested in vitro with DNA of certain Babesia species that infect dogs. The hsp70 gene is a precise molecular marker for Babesia phylogeny, especially species that infect dogs.</abstract><cop>Netherlands</cop><pub>Elsevier GmbH</pub><pmid>29409719</pmid><doi>10.1016/j.ttbdis.2018.01.013</doi><tpages>7</tpages></addata></record> |
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| ispartof | Ticks and tick-borne diseases, 2018-03, Vol.9 (3), p.556-562 |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Animals Babesia - chemistry Babesia - genetics Babesia - isolation & purification Babesiosis Babesiosis - blood Babesiosis - diagnosis Babesiosis - epidemiology Babesiosis - parasitology Brazil - epidemiology DNA Primers - genetics DNA, Protozoan - genetics Dog Diseases - diagnosis Dog Diseases - epidemiology Dog Diseases - parasitology Dogs Heat shock protein 70 kDa HSP70 Heat-Shock Proteins - genetics Molecular Diagnostic Techniques - methods Phylogeny Real-time polymerase chain reaction Real-Time Polymerase Chain Reaction - methods RNA, Ribosomal, 18S - genetics |
| title | Comparison of heat shock protein 70 kDa and 18S rDNA genes for molecular detection and phylogenetic analysis of Babesia vogeli from whole blood of naturally infected dogs |
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