Comparison of Immunomagnetic Bead Separation-Immunosensor Detection and Nested-PCR Methods for Detecting Mycobacterium avium Subspecies paratuberculosis in Cattle Feces.

Publisher: Wiley Country of Publication: United States NLM ID: 8801384 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1098-2825 (Electronic) Linking ISSN: 08878013 NLM ISO Abbreviation: J Clin Lab Anal Subsets: MEDLINE

Publication: Hoboken, N.J. : Wiley
Original Publication: New York : Alan R. Liss, Inc., c1987-

Mycobacterium avium subsp. paratuberculosis*/isolation & purification ; Mycobacterium avium subsp. paratuberculosis*/genetics ; Feces*/microbiology ; Immunomagnetic Separation*/methods ; Paratuberculosis*/diagnosis ; Paratuberculosis*/microbiology ; Polymerase Chain Reaction*/methods ; Polymerase Chain Reaction*/veterinary ; Cattle Diseases*/diagnosis ; Cattle Diseases*/microbiology ; Biosensing Techniques*/methods; Animals ; Cattle ; Sensitivity and Specificity

Background: Johne's disease, also known as paratuberculosis, is a chronic granulomatous enteritis disease that affects ruminants worldwide.
Objective: The objective of this study was to assess the effectiveness of the immunomagnetic bead separation-immunosensor (IMB-IS) detection method compared to Nested-PCR for identifying Mycobacterium avium subspecies paratuberculosis (MAP) infection in cattle feces samples.
Methods: Ninety rectal fecal samples were collected from selected cattle, comprising 59 serum-positive and 31 serum-negative cases based on serum ELISA. Following DNA extraction, nested-PCR was conducted using the IS900 primer sequence targeting the MAP-specific gene. Immunomagnetic bead (IMB) nanoparticles were synthesized by purifying hyperimmune donkey IgG through affinity chromatography and then conjugating it to Fe nanoparticles. Rhodamine-B hydrazone immunosensor (IS) was synthesized and conjugated to hyperimmune rabbit IgG. The synthesized IMB and IS were used to identify MAP in cattle fecal samples.
Results: The results of this study revealed that of the 90 stool samples tested using the nested-PCR method, 62 samples (68.88%) were positive, while 28 samples (31.12%) were negative. In the IMB-IS test based on optical density (OD), 64 samples were positive (71.1%), while 26 samples were negative (28.8%). This test exhibited a sensitivity of 100%, specificity of 92.85%, and an overall test accuracy of 97.77%.
Conclusion: Given the considerations of cost, time, positive and negative predictive values, and acceptable accuracy of the IMB-IS test, it is recommended for evaluation in screening and epidemiological studies.
(© 2025 The Author(s). Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Appl Environ Microbiol. 1998 Sep;64(9):3153-8. (PMID: 9726853)
Biomed Res Int. 2017;2017:5869854. (PMID: 28642876)
J Dairy Sci. 2021 Jun;104(6):6944-6960. (PMID: 33814150)
Vet Microbiol. 2013 Dec 27;167(3-4):725-8. (PMID: 24100005)
Braz J Microbiol. 2016 Apr-Jun;47(2):506-12. (PMID: 26991290)
Med Vet Entomol. 2005 Dec;19(4):360-6. (PMID: 16336300)
Appl Environ Microbiol. 2010 Nov;76(22):7550-8. (PMID: 20851966)
Int J Food Microbiol. 2006 Aug 1;110(3):201-8. (PMID: 16814891)
Vet J. 2014 Jan;199(1):138-42. (PMID: 24280588)
Chem Soc Rev. 2009 Aug;38(8):2410-33. (PMID: 19623358)
Vet Microbiol. 2011 Dec 29;154(1-2):197-201. (PMID: 21775077)
J Clin Microbiol. 1996 Mar;34(3):516-9. (PMID: 8904405)
Clin Microbiol Rev. 1994 Jan;7(1):43-54. (PMID: 8118790)
Int J Food Microbiol. 2008 Dec 10;128(2):250-7. (PMID: 18824269)
Prev Vet Med. 2018 Apr 1;152:74-80. (PMID: 29559108)
Bioconjug Chem. 2009 Oct 21;20(10):1966-74. (PMID: 19751063)
BMC Microbiol. 2002 Jun 26;2:15. (PMID: 12097144)
Clin Vaccine Immunol. 2006 Oct;13(10):1125-30. (PMID: 16928884)
Clin Vaccine Immunol. 2008 Feb;15(2):227-34. (PMID: 18077613)
J Appl Microbiol. 2010 May;108(5):1485-93. (PMID: 19912432)
Anal Biochem. 2010 Mar 1;398(1):120-2. (PMID: 19903448)
Indian J Exp Biol. 2007 Oct;45(10):843-52. (PMID: 17948732)
Vet Microbiol. 2008 Jun 22;129(3-4):217-35. (PMID: 18255239)
Int J Food Microbiol. 1999 Jan 12;46(1):37-44. (PMID: 10050683)
Anal Chem. 2008 Dec 15;80(24):9612-21. (PMID: 19007241)
Prev Vet Med. 2015 Sep 1;121(1-2):49-55. (PMID: 26092721)
J Zhejiang Univ Sci B. 2017 Oct.;18(10):845-853. (PMID: 28990375)
J Appl Microbiol. 2008 Jul;105(1):227-35. (PMID: 18284481)
J Dairy Sci. 2008 Jul;91(7):2620-7. (PMID: 18565921)
J Agric Food Chem. 2009 Jan 28;57(2):517-24. (PMID: 19154162)
Front Vet Sci. 2021 Feb 03;8:615029. (PMID: 33614761)
Biosens Bioelectron. 2012 Jun-Jul;36(1):135-41. (PMID: 22560440)
J Food Prot. 2001 Nov;64(11):1744-50. (PMID: 11726153)
Bioconjug Chem. 2009 Jul;20(7):1349-55. (PMID: 19534519)
J Dairy Sci. 2012 May;95(5):2734-9. (PMID: 22541503)
Anal Chem. 2009 Jul 15;81(14):5637-45. (PMID: 19522527)
Sci Rep. 2014 Oct 28;4:6794. (PMID: 25348724)
Vet Microbiol. 2000 Dec 20;77(3-4):369-78. (PMID: 11118722)
J Clin Microbiol. 2004 Mar;42(3):1075-81. (PMID: 15004056)
Can J Vet Res. 2004 Oct;68(4):302-8. (PMID: 15581226)
Clin Diagn Lab Immunol. 1998 Jul;5(4):446-51. (PMID: 9665946)

SCU.VP1402.12470 Shahid Chamran University of Ahvaz

Keywords: Mycobacterium avium subspecies paratuberculosis; immunomagnet; immunosensor; nested‐PCR; paratuberculosis

Date Created: 20250319 Date Completed: 20250410 Latest Revision: 20250412

20250414

PMC11981956

10.1002/jcla.70009

40105275