The Ability of primer IS900 and F57 to detect mycobacterium avium subspecies paratuberculosis by conventional PCR

Widagdo Sri Nugrobo, . and Mirnawati Sudarwanto, . and Denny Widaya Lukman, . and Surachmi Setyaningsih, . and Abdulwahed Ahmed Hassan, . and Ewald Usleber, . (2009) The Ability of primer IS900 and F57 to detect mycobacterium avium subspecies paratuberculosis by conventional PCR. [Proceedings Paper]

Johne's disease JD is chronic granulomatous enteritis in ruminants which infected by Mycobacterium avium subspecies paratuberculosis MAP and this inflammatory disorder shows many similarities to Crohn's disease CD in human. Few researches conducted in Europe USA and Australia showed the relationship among MAP CD JD and dairy products. The detection method of MAP was developed by using polymerase chain reaction PCR. The most popular primer used in this method was IS 900 but in the last few years some researchers developed primer F57 to improve its accuracy. Some researchers showed that a variety of performance of both primers on the MAP depends on the samples and the amount of bacteria in it. The aims ofthis study were to evaluate sensitivity and specificity ofthe primer IS 900 and F57 to detect MAP by conventional PCR. Primers configurations oflS900 described by Bull 2003 are TJ I: 5'-GCT GAT CGC CTTGCTCAT-3' andTJ2: 5'-CGG GAG TTTGGTAGC CAGTA-3'. And oligonucloetide of primer F57 are F57/R57F57:5'-CCT GTC TAA TTC GAT CAC GGA CTA GA-3' and R57:5'-TCA GCT ATT GGT GTA CCG AAT GT-3' Vansnick et al. 2004. Sensitivity test was done by using serial dilutions of MAP reference from 10-10' celllmi in PBS Tween 0.5. Every dilution suspension of MAP was inoculated into herrold's egg yolk medium enrichment with mycobactin J as a gold standard. Specificity evaluation was done by comparing the MAP to Mycobacterium avium subspecies avium MAV Escherichia coli Listeria ivanovii Pseudomonas aeruginusa Streptococcus agalactiae Klebsiella pneumoniae Staphylococcus hyicus Streptococcus infatis and Listeria monocytogenes. The DNA extractions of all isolates were conducted by using DNeasy Tissue Kit Qiagen Hilden Germany. Amplification condition for PCR were 1 cycle at 94C for 10 minutes 40 cycles at 94C for 1 minute 58C for 1 minute and 72 C for 3 minutes and 1 cycle at 72 C for 7 minutes. Followed by electrophoresis the gels were stained for 5 min with 5ul/ml ethidium bromide solution Sigma Tautkirchen Germany and it was visualized under a UV 245 nm trans-illuminator Biorad and use marker of 100 bp DNA ladder MBI Fermentas St. Leon-Rot Germany. The result showed that both primers specific for MAP DNA. The primer IS 900 detected MAP DNA from 104 cell/ml and primer F57 detected at 10-3 cell/ml.

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Abstract

Johne's disease JD is chronic granulomatous enteritis in ruminants which infected by Mycobacterium avium subspecies paratuberculosis MAP and this inflammatory disorder shows many similarities to Crohn's disease CD in human. Few researches conducted in Europe USA and Australia showed the relationship among MAP CD JD and dairy products. The detection method of MAP was developed by using polymerase chain reaction PCR. The most popular primer used in this method was IS 900 but in the last few years some researchers developed primer F57 to improve its accuracy. Some researchers showed that a variety of performance of both primers on the MAP depends on the samples and the amount of bacteria in it. The aims ofthis study were to evaluate sensitivity and specificity ofthe primer IS 900 and F57 to detect MAP by conventional PCR. Primers configurations oflS900 described by Bull 2003 are TJ I: 5'-GCT GAT CGC CTTGCTCAT-3' andTJ2: 5'-CGG GAG TTTGGTAGC CAGTA-3'. And oligonucloetide of primer F57 are F57/R57F57:5'-CCT GTC TAA TTC GAT CAC GGA CTA GA-3' and R57:5'-TCA GCT ATT GGT GTA CCG AAT GT-3' Vansnick et al. 2004. Sensitivity test was done by using serial dilutions of MAP reference from 10-10' celllmi in PBS Tween 0.5. Every dilution suspension of MAP was inoculated into herrold's egg yolk medium enrichment with mycobactin J as a gold standard. Specificity evaluation was done by comparing the MAP to Mycobacterium avium subspecies avium MAV Escherichia coli Listeria ivanovii Pseudomonas aeruginusa Streptococcus agalactiae Klebsiella pneumoniae Staphylococcus hyicus Streptococcus infatis and Listeria monocytogenes. The DNA extractions of all isolates were conducted by using DNeasy Tissue Kit Qiagen Hilden Germany. Amplification condition for PCR were 1 cycle at 94C for 10 minutes 40 cycles at 94C for 1 minute 58C for 1 minute and 72 C for 3 minutes and 1 cycle at 72 C for 7 minutes. Followed by electrophoresis the gels were stained for 5 min with 5ul/ml ethidium bromide solution Sigma Tautkirchen Germany and it was visualized under a UV 245 nm trans-illuminator Biorad and use marker of 100 bp DNA ladder MBI Fermentas St. Leon-Rot Germany. The result showed that both primers specific for MAP DNA. The primer IS 900 detected MAP DNA from 104 cell/ml and primer F57 detected at 10-3 cell/ml.

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Item Type:	Proceedings Paper
Additional Information:	3 ill.
AGROVOC Term:	Polymerase chain reaction
AGROVOC Term:	Mycobacterium avium subsp. paratuberculosis
AGROVOC Term:	Johne's disease
AGROVOC Term:	Chronic granulomatous diseas
AGROVOC Term:	Dairy products
Geographical Term:	MALAYSIA
Depositing User:	Ms. Norfaezah Khomsan
Last Modified:	24 Apr 2025 05:27
URI:	http://webagris.upm.edu.my/id/eprint/15772

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