Comparative evaluation of various in-house protocols on diagnostic performance for paratuberculosis IS900 PCR.
Fecal DNA isolation
Fecal IS900 PCR
PCR Master mixes
Paratuberculosis
Journal
Molecular biology reports
ISSN: 1573-4978
Titre abrégé: Mol Biol Rep
Pays: Netherlands
ID NLM: 0403234
Informations de publication
Date de publication:
Jan 2023
Jan 2023
Historique:
received:
08
08
2022
accepted:
23
09
2022
pubmed:
14
11
2022
medline:
1
2
2023
entrez:
13
11
2022
Statut:
ppublish
Résumé
Paratuberculosis is a worldwide endemic infectious disease of ruminants that results in high economic losses. Public health concerns are also being raised with human Crohn's disease. Therefore, control is becoming priority for governments. Control is largely dependent on "Test and Cull" or "Test and Segregate" policy. Hence, it is critical to assure the infection before making the decision. Commercial kits are costly especially in view of resource limited areas. Present study analyzed the performance various in house DNA isolation methods and PCR master mix combinations to optimize a protocol for confirmation of paratuberculosis bacilli shedding in feces. Present study included five protocols of fecal DNA isolation (chemical, bio-chemical, physio-chemical and physical) and three reaction mixes (based on Qiagen, Genei and Thermo 2X master mixes) in nine different combinations using additives and tested their performance for IS900 PCR. Spiked fecal samples were used to select the best combination of DNA isolation method and PCR master mix (PRM). Selected combination was used to test reference (positive and negative) fecal samples and field samples. Findings revealed that combination physical method of DNA isolation and Genei based PRM (with additives; betaine DMSO and BSA) had lowest limit of detection. Sensitivity was 83% and specificity was 100% in comparison to fecal culture. High prevalence (23%) was reported for paratuberculosis on field samples. Optimized protocol has acceptable sensitivity and can easily be adopted in resource-limited laboratories. High prevalence of paratuberculosis needs immediate implementation of the control strategies.
Sections du résumé
BACKGROUND
BACKGROUND
Paratuberculosis is a worldwide endemic infectious disease of ruminants that results in high economic losses. Public health concerns are also being raised with human Crohn's disease. Therefore, control is becoming priority for governments. Control is largely dependent on "Test and Cull" or "Test and Segregate" policy. Hence, it is critical to assure the infection before making the decision. Commercial kits are costly especially in view of resource limited areas. Present study analyzed the performance various in house DNA isolation methods and PCR master mix combinations to optimize a protocol for confirmation of paratuberculosis bacilli shedding in feces.
METHODS AND RESULTS
RESULTS
Present study included five protocols of fecal DNA isolation (chemical, bio-chemical, physio-chemical and physical) and three reaction mixes (based on Qiagen, Genei and Thermo 2X master mixes) in nine different combinations using additives and tested their performance for IS900 PCR. Spiked fecal samples were used to select the best combination of DNA isolation method and PCR master mix (PRM). Selected combination was used to test reference (positive and negative) fecal samples and field samples. Findings revealed that combination physical method of DNA isolation and Genei based PRM (with additives; betaine DMSO and BSA) had lowest limit of detection. Sensitivity was 83% and specificity was 100% in comparison to fecal culture. High prevalence (23%) was reported for paratuberculosis on field samples.
CONCLUSION
CONCLUSIONS
Optimized protocol has acceptable sensitivity and can easily be adopted in resource-limited laboratories. High prevalence of paratuberculosis needs immediate implementation of the control strategies.
Identifiants
pubmed: 36371554
doi: 10.1007/s11033-022-07984-z
pii: 10.1007/s11033-022-07984-z
doi:
Substances chimiques
DNA
9007-49-2
DNA, Bacterial
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
943-947Subventions
Organisme : Biotechnology Industry Research Assistance Council
ID : BT/CRS0226/CRS-11/17
Organisme : Indian Council of Medical Research
ID : Indian Council of Medical Research
Informations de copyright
© 2022. The Author(s), under exclusive licence to Springer Nature B.V.
Références
Gilardoni LR, Paolicchi FA, Mundo SL (2012) Bovine paratuberculosis: a review of the advantages and disadvantages of different diagnostic tests. Rev Argent Microbiol 44:201–215
Fechner K, Dreymann N, Schimkowiak S, Czerny CP, Teitzel J (2019) Efficacy of dairy on-farm high-temperature, short-time pasteurization of milk on the viability of Mycobacterium avium ssp. paratuberculosis. J Dairy Sci 102:11280–11290. https://doi.org/10.3168/jds.2019-16590
doi: 10.3168/jds.2019-16590
Davis WC, Kuenstner JT, Singh SV (2017) Resolution of Crohn’s (Johne’s) disease with antibiotics: what are the next steps? Expert Rev Gastroenterol Hepatol 11:393–396. https://doi.org/10.1080/17474124.2017.1300529
doi: 10.1080/17474124.2017.1300529
Sohal JS, Singh SV, Singh B, Thakur S, Aseri GK, Jain N, Jayaraman S, Yadav P, Khare N, Gupta S, Chaubey KK, Dhama K (2015) Control of Paratuberculosis: Opinions and Practices. Adv Anim Vet Sci 3:156–163. https://doi.org/10.14737/journal.aavs/2015/3.3.156.163
doi: 10.14737/journal.aavs/2015/3.3.156.163
Whittington R, Donat K, Weber MF (2019) Control of paratuberculosis: who, why and how. A review of 48 countries. BMC Vet Res 2019:198. https://doi.org/10.1186/s12917-019-1943-4
Sohal JS, Singh SV, Subhodh S, Singh AV, Singh PK, Sheoran N, Sandhu K, Narayansamy K, Maitra A (2007) Mycobacterium avium subspecies paratuberculosis diagnosis and strain typing- present status and future developments. Indian J Exp Biol 45:843–852
Karunanathie H, Kee PS, Ng SF, Kennedy MA, Chua EW (2022) PCR enhancers: Types, mechanisms, and applications in long-range PCR. Biochimie 197:130–143
doi: 10.1016/j.biochi.2022.02.009
Singh SV, Singh AV, Singh R et al (2007) Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal-PCR for the diagnosis of bovine Johne’s disease (BJD) in India. Comp Immunol Microbiol Infect Dis 30:175–186. https://doi.org/10.1016/j.cimid.2006.12.002
doi: 10.1016/j.cimid.2006.12.002
Bag S, Saha B, Mehta O et al (2016) An improved method for high quality metagenomics DNA extraction from human and environmental samples. Sci Rep 6:26775
doi: 10.1038/srep26775
Stabel JR, Bosworth TL, Kirkbride TA, Forde RL, Whitlock RH (2004) A simple, rapid, and effective method for the extraction of Mycobacterium paratuberculosis DNA from fecal samples for polymerase chain reaction. J Vet Diagn Invest 16:22–30. https://doi.org/10.1177/104063870401600105
doi: 10.1177/104063870401600105
Haghkhah M, Derakhshandeh A, Jamshidi R, Moghiseh A, Karimaghaei N, Ayaseh M, Mostafaei M (2015) Detection of Mycobacterium avium subspecies paratuberculosis infection in two different camel species by conventional and molecular techniques. Vet Res Forum 6:337–341
Lorenz TC (2012) Polymerase chain reaction: basic protocol plus troubleshooting and optimization strategies. J Vis Exp 63:3998. https://doi.org/10.3791/3998
doi: 10.3791/3998
Varadharajan B, Parani M (2021) DMSO and betaine significantly enhance the PCR amplification of ITS2 DNA barcodes from plants. Genome 64:165–171. https://doi.org/10.1139/gen-2019-0221
doi: 10.1139/gen-2019-0221
Musso M, Bocciardi R, Parodi S, Ravazzolo R, Ceccherini I (2006) Betaine, dimethyl sulfoxide, and 7-deaza-dGTP, a powerful mixture for amplification of GC-rich DNA sequences. J Mol Diagn 8:544–550. https://doi.org/10.2353/jmoldx.2006.060058
doi: 10.2353/jmoldx.2006.060058
Woide D, Zink A, Thalhammer S (2010) Technical note: PCR analysis of minimum target amount of ancient DNA. Am J Phys Anthropol 142:321–327. https://doi.org/10.1002/ajpa.21268
doi: 10.1002/ajpa.21268
Farell EM, Alexandre G (2012) Bovine serum albumin further enhances the effects of organic solvents on increased yield of polymerase chain reaction of GC-rich templates. BMC Res Notes 5:257. http://www.biomedcentral.com/1756-0500/5/257
doi: 10.1186/1756-0500-5-257
Sharma S, Gautam AK, Singh SV, Chaubey KK, Rose MK, Bangar Y, Gururaj K (2021) In vivo kinetics of peripheral cellular immune responses in Mycobacterium avium subspecies paratuberculosis (MAP) infected and vaccinated goats. Comp Immunol Microbiol Infect Dis 79:101710
doi: 10.1016/j.cimid.2021.101710
Bhutediya JM, Dandapat P, Chakrabarty A et al (2017) Prevalence of paratuberculosis in organized and unorganized dairy cattle herds in West Bengal, India. Vet World 10:574–579. https://doi.org/10.14202/vetworld.2017.574-579
doi: 10.14202/vetworld.2017.574-579
Barad DB, Chandel BS, Dadawala AI, Chauhan HC, Kher HS, Shroff S, Bhagat AG, Singh SV, Singh PK, Singh AV, Sohal JS, Gupta S, Chaubey KK, Chakraborty S, Tiwari R, Deb R, Dhama K (2014) Incidence of Mycobacterium avium subspecies paratuberculosis in Mehsani and Surti Goats of Indian origin using multiple diagnostic tests. J Biol Sci 14:124–133
doi: 10.3923/jbs.2014.124.133
Singh SV, Singh PK, Singh AV et al (2014) ‘Bio-load’ and bio-type profiles of Mycobacterium avium subspecies paratuberculosis infection in the domestic livestock population endemic for Johne’s disease: a survey of 28 years (1985–2013) in India. Transbound Emerg Dis 61:43–55. https://doi.org/10.1111/tbed.12216
doi: 10.1111/tbed.12216
Garg R, Patil PK, Singh SV, Sharma S, Gandham RK, Singh AV, Filia G, Singh PK, Jayaraman S, Gupta S, Chaubey KK, Tiwari R, Saminathan M, Dhama K, Sohal JS (2015) Comparative evaluation of different test combinations for diagnosis of Mycobacterium avium subspecies paratuberculosis infecting dairy herds in India. BioMed Res Int 2015:983978. https://doi.org/10.1155/2015/983978