Document Type : Research Paper
Authors
Department of Microbiology; College of Veterinary Medicine; University of Basrah, Basrah, Iraq.
10.29079/qjvms.2023.179348
Abstract
This study aimed to compare the efficiency of conventional microbiological, Miniaturizekit GP24 and molecular technique by using PCR of nuc gene in the identification of S. aureus.Also to evaluate the efficiency of the tube coagulase and DNase activity tests. 104 of suspected S.aureus was isolated from different cattle samples, (68.3%) and (75%) were positive forcoagulase and DNase tests, respectively. The sensitivity and specificity of coagulase andDNase were 92.5%, 75.7% and 79.1% , 32.4% respectively. The identification rate of S.aureus by GP24 kit and PCR detection using nuc gene was 68.26% and 64.42% respectively.Out of 104(31.7%) of isolates were identified as coagulase-negative staphylococci by usingGP24 kit. Antimicrobials susceptibility assays of 67 S. aureus isolates revealed that, theThe highest resistance rates were against penicillin (97%) followed by tetracycline (25.4%).While all isolates were found to be highly sensitive toward Imipenem, vancomycin, andchloramphenicol (100%) for each. In conclusion, the coagulase and conventionalmicrobiological techniques have more reliable results in comparison with the DNase test forthe identification of S. aureus. On other hand, the results of the GP24 kit and coagulase tube testwere similar in sensitivity and specificity.
Keywords
1. Markey BK, Leonard FC, Archambault M, Cullinane A, Maguire D. Clinical veterinary microbiology. 2nd ed. Staphylococcus spp. 2013:105-20.
2. Eslampour MA, Hovareshti P, Feizabadi MM. Molecular characterization of Staphylococcus aureus isolated from bovine mastitis in Iran. Vet Microbiol. 2009;39:207-8.https://doi.org/10.1016/j.vetmic.2009.05.001
3. Hasman H, Moodley A, Guardabassi L. Spa type distribution in Staphylococcus aureus originating from pigs, cattle and poultry. Vet Microbiol. 2010;141:326-31.https://doi.org/10.1016/j.vetmic.2009.09.025
4. Amagliani G, Glammarini C, Omicciolo E, Brandi G, Magnami M. Food Control. 2007;18:1137-42.https://doi.org/10.1016/j.foodcont.2006.06.012
5. Sommer LY, Kashi J. Food Prot. 2003;66:1658-65.https://doi.org/10.4315/0362-028X-66.9.1658
6. Ryan KJ, Ray CG. Sherris Medical microbiology: An Introduction to Infectious Diseases. 4th ed. McGraw Hill Publishers; 2004.
7. Windria S, Widianingrum DC, Salasia SIO. Identification of Staphylococcus aureus and coagulase-negative staphylococci isolates from mastitis milk of Etawa crossbred goat. Res J Microbiol. 2016;11:11-9.https://doi.org/10.3923/jm.2016.11.19
8. Zhang K, Sparling J, Chow BL, Elsayed S, Hussain Z, Church DL. New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. J Clin Microbiol. 2004;42(11):4947-55.https://doi.org/10.1128/JCM.42.11.4947-4955.2004
9. Baldhar CN, Chilgunde SN, Gaikwad RV, Smad A, Roy S. Current trends in diagnosis of mastitis in Veterinary practice. J Remt Vet Corp. 2005;44(3):133-40.
10. Quinn PJ, Markey BK, Leonard FC, Fitzpatrick ES, Fanning S, Hartigan PJ. Veterinary microbiology and microbial disease: Staphylococcus species. 2nd ed. Wiley-Blackwell; 2011:179-87.
11. Tucker PW, Hazen EE, Cotton FA. Staphylococcal nuclease reviewed: a prototypic study in contemporary enzymology. I. Isolation, physical and enzymatic properties. Mol Cell Biochem. 1978;22:67-77.https://doi.org/10.1007/BF00496235
12. Zhang K, Sparling J, Chow BL, Elsayed S, Hussain Z, Church DL. New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. J Clin Microbiol. 2004;42(11):4947-55.https://doi.org/10.1128/JCM.42.11.4947-4955.2004
13. Macfaddin JF. Biochemical tests for identification of medical bacteria. 3rd ed. Lippincott Williams and Wilkins USA; 2000.
14. Longboot W, Chomvarin C, Engchanil C, Chaimanee P. Multiplex PCR for detection of superantigenic toxin genes in methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolated from patients and carriers of a hospital in northeast Thailand. Southeast Asian J Trop Med Public Health. 2013;44:660-71.
15. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests-Eighth Edition; Approved Standard M2-A8. NCCLS, Wayne, PA, USA; 2013.
16. Nahavandi K. Calculating Sensitivity, Specificity and Predictive Values for Medical Diagnostic Tests. Gene Cell Tissue. 2018;5(2):e80270.https://doi.org/10.5812/gct.80270
17. Kateete DP, Kimani CN, Katabazi FA, Okeng A, Okee MS, Nanteza A, et al. Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test. Ann Clin Microbiol Antimicrob. 2010;9:23.https://doi.org/10.1186/1476-0711-9-23
18. Aboud WA. Molecular Detection of Vancomycin and Methicillin-Resistant Genes of Staphylococcus aureus Isolated from Animal and Human Specimens. MSc Thesis, College of Veterinary Medicine, University of Basrah; 2019.
19. Mahdi ASH. Molecular Detection of Superantigens in Staphylococcus aureus Isolated from Human, Cattle and Buffalo Milk Samples. MSc Thesis, College of Veterinary Medicine, University of Basrah; 2019.
20. Singh RS, Kumar R, Yadav BR. Distribution of pathogenic factors in Staphylococcus aureus strains isolated from intra-mammary infections in cattle and buffaloes. Indian J Biotechnol. 2011;10:410-6.
21. Turkyilmaz S, Kaya O. Determination of some virulence factors in Staphylococcus spp. isolated from various clinical samples. Turk J Vet Anim Sci. 2005;30:127-32.
22. David FT, Taku A, Bhat MA, Badroo M, Sofi TM. Molecular typing of Staphylococcus aureus based on coagulase gene. Vet World. 2018;11:2231-6.https://doi.org/10.14202/vetworld.2018.423-430
23. Pumipuntu N, Kulpeanprasit S, Santajit S, Tunyong W, Kong-ngoen T, Hinthong W, et al. Screening method for Staphylococcus aureus identification in subclinical bovine mastitis from dairy farms. Vet World. 2017;10:2231-6.https://doi.org/10.14202/vetworld.2017.721-726
24. Gianneechini R, Conch C, Rivero R, Delucci I, Moreno Lopez J. Occurrence of Clinical and Subclinical Mastitis in Dairy Herds in the West Littoral Region in Uruguay. Acta Vet Scand. 2002;43:221-30.https://doi.org/10.1186/1751-0147-43-221
25. Myllys V, Asplund K, Brofeldt E, Hirvela-Koski V, Honkanen-Buzalaske T, Junttila J, et al. Bovine mastitis in Finland in 1988 and 1995-changes in prevalence and antimicrobial resistance. Acta Vet Scand. 1998;39:119-26.https://doi.org/10.1186/BF03547813
26. Pyorala S, Taponen S. Coagulase-negative staphylococci emerging mastitis pathogens. Vet Microbiol. 2009;134:3-8.https://doi.org/10.1016/j.vetmic.2008.09.015
27. AL-Edany A, Khudor M, AL-Mousawi Kh. Comparison of three indirect tests for the diagnosis of bovine subclinical mastitis caused by coagulase-negative staphylococci with their susceptibility to seven antibiotics. Bas J Vet Res. 2012;11(1).https://doi.org/10.33762/bvetr.2012.54759
28. Foster TJ. Immune evasion by staphylococci. Nat Rev Microbiol. 2005;3:948-58.https://doi.org/10.1038/nrmicro1289
29. Palomares C, Torres MJ, Torres A, Aznar J, Palomares JC. Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR. Diagn Microbiol Infect Dis. 2003;45:183-9.https://doi.org/10.1016/S0732-8893(02)00542-4
30. Costa AM, Kay I, Palladino S. Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction. Diagn Microbiol Infect Dis. 2005;51:13-7.https://doi.org/10.1016/j.diagmicrobio.2004.08.014
31. Asfour HAE, Darwish SF. Phenotypic and genotypic detection of both mecA and blaZ genes mediated β-lactam resistance in Staphylococcus strains isolated from bovine mastitis. Glob Vet. 2011;6:39-50.
32. Hammad AM. Microbiological studies on raw milk and some dairy products. MSc Thesis, Minufiya Univ., Egypt; 2004.
33. Rall VLM, Vieira FP, Rall R, Vieitis RL, Fernandes A, Candeias JMG, et al. PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk. Vet Microbiol. 2008;132(3):408-13.https://doi.org/10.1016/j.vetmic.2008.05.011
34. Jahan M, Rahman M, Parvej M, Chowdhury S, Haque M, Talukder M, et al. Isolation and characterization of S. aureus from raw cow milk in Bangladesh. J Adv Vet Anim Res. 2015;2(1):49-59.https://doi.org/10.5455/javar.2015.b47
35. Liu H, Li S, Meng L, Dong L, Zhao S, Lan X, et al. Prevalence, antimicrobial susceptibility, and molecular characterization of S. aureus isolated from dairy herds in northern China. J Dairy Sci. 2017;100(5):340-7.https://doi.org/10.3168/jds.2017-13370
36. Momtaz H, Tajbakhsh E, Rahimi E, Momeni M. Coagulase gene polymorphism of S. aureus isolated from clinical and sub-clinical bovine mastitis in Isfahan and Chaharmahal va Bakhtiari provinces of Iran. Comp Clin Pathol. 2011;20(5):519-22.https://doi.org/10.1007/s00580-010-1029-y
37. Vintov J, Aarestrup FM, Zinn CE, Olsen JE. Association between phage types and antimicrobial resistance among bovine Staphylococcus aureus from 10 countries. Vet Microbiol. 2003;95:133-47.https://doi.org/10.1016/S0378-1135(03)00156-1
38. Idbeis HI. Molecular Analysis of Staphylococcus aureus Isolated From Mastitis and Study Their Biofilm Activity against Cinnamon Oil. PhD thesis, College of Veterinary Medicine, University of Basrah; 2019.
39. Alian F, Rahimi E, Shakerian A, Momtaz H, Riahi M, Momeni M. Antimicrobial resistance of Staphylococcus aureus isolated from bovine, sheep and goat raw milk. Glob Vet. 2012;8:111-4.
40. Al-Iedani AA. Phenotypic study on the capacity of biofilm production in S. aureus isolated from bovine subclinical mastitis and their impact on resistance to antimicrobials. Basrah J Vet Res. 2016;15(2):111-27.https://doi.org/10.33762/bvetr.2016.124293
41. Aliyu Y, Reuben C, Sani A, Salawu E. Occurrence and antibiogram of S. aureus isolated from locally-pasteurised cow milk (kindirmo) sold in parts of Nasarawa Town, Nasarawa State, Nigeria. Microbiol Res J Int. 2018;23(4):1-11.https://doi.org/10.9734/MRJI/2018/40294
42. Michael CA, Domine-Howes D, Labbate M. The antimicrobial resistance crisis: causes, consequences, and management. Front Public Health. 2014;2.https://doi.org/10.3389/fpubh.2014.00145
43. Aldhar CN, Chilgunde SN, Gaikwad RV, Smad A, Roy S. Current trends in diagnosis of mastitis in Veterinary practice. J Remt Vet Corp. 2005;44(3):133-40.
44. Yadav R, Sharma SK, Yadav J, Nathawar P, Kataria AK. Phenotypic and genotypic characterization of Staphylococcus aureus of mastitic milk origin from cattle and buffalo for some virulence properties. J Pure Appl Microbiol. 2009;3(1):353-6.
45. Davis TE, Fuller DD, Aeschleman EC. Rapid, direct identification of Staphylococcus aureus and Streptococcus pneumoniae from blood cultures using commercial immunological kits and modified conventional tests. Diagn Microbiol Infect Dis. 1992;15:295-300.https://doi.org/10.1016/0732-8893(92)90014-K
46. Foster TJ. Immune evasion by staphylococci. Nat Rev Microbiol. 2005;3:948-58.https://doi.org/10.1038/nrmicro1289
47. Palomares C, Torres MJ, Torres A, Aznar J, Palomares JC. Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR. Diagn Microbiol Infect Dis. 2003;45:183-9.https://doi.org/10.1016/S0732-8893(02)00542-4
2. Eslampour MA, Hovareshti P, Feizabadi MM. Molecular characterization of Staphylococcus aureus isolated from bovine mastitis in Iran. Vet Microbiol. 2009;39:207-8.https://doi.org/10.1016/j.vetmic.2009.05.001
3. Hasman H, Moodley A, Guardabassi L. Spa type distribution in Staphylococcus aureus originating from pigs, cattle and poultry. Vet Microbiol. 2010;141:326-31.https://doi.org/10.1016/j.vetmic.2009.09.025
4. Amagliani G, Glammarini C, Omicciolo E, Brandi G, Magnami M. Food Control. 2007;18:1137-42.https://doi.org/10.1016/j.foodcont.2006.06.012
5. Sommer LY, Kashi J. Food Prot. 2003;66:1658-65.https://doi.org/10.4315/0362-028X-66.9.1658
6. Ryan KJ, Ray CG. Sherris Medical microbiology: An Introduction to Infectious Diseases. 4th ed. McGraw Hill Publishers; 2004.
7. Windria S, Widianingrum DC, Salasia SIO. Identification of Staphylococcus aureus and coagulase-negative staphylococci isolates from mastitis milk of Etawa crossbred goat. Res J Microbiol. 2016;11:11-9.https://doi.org/10.3923/jm.2016.11.19
8. Zhang K, Sparling J, Chow BL, Elsayed S, Hussain Z, Church DL. New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. J Clin Microbiol. 2004;42(11):4947-55.https://doi.org/10.1128/JCM.42.11.4947-4955.2004
9. Baldhar CN, Chilgunde SN, Gaikwad RV, Smad A, Roy S. Current trends in diagnosis of mastitis in Veterinary practice. J Remt Vet Corp. 2005;44(3):133-40.
10. Quinn PJ, Markey BK, Leonard FC, Fitzpatrick ES, Fanning S, Hartigan PJ. Veterinary microbiology and microbial disease: Staphylococcus species. 2nd ed. Wiley-Blackwell; 2011:179-87.
11. Tucker PW, Hazen EE, Cotton FA. Staphylococcal nuclease reviewed: a prototypic study in contemporary enzymology. I. Isolation, physical and enzymatic properties. Mol Cell Biochem. 1978;22:67-77.https://doi.org/10.1007/BF00496235
12. Zhang K, Sparling J, Chow BL, Elsayed S, Hussain Z, Church DL. New quadriplex PCR assay for detection of methicillin and mupirocin resistance and simultaneous discrimination of Staphylococcus aureus from coagulase-negative staphylococci. J Clin Microbiol. 2004;42(11):4947-55.https://doi.org/10.1128/JCM.42.11.4947-4955.2004
13. Macfaddin JF. Biochemical tests for identification of medical bacteria. 3rd ed. Lippincott Williams and Wilkins USA; 2000.
14. Longboot W, Chomvarin C, Engchanil C, Chaimanee P. Multiplex PCR for detection of superantigenic toxin genes in methicillin-sensitive and methicillin-resistant Staphylococcus aureus isolated from patients and carriers of a hospital in northeast Thailand. Southeast Asian J Trop Med Public Health. 2013;44:660-71.
15. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Disk Susceptibility Tests-Eighth Edition; Approved Standard M2-A8. NCCLS, Wayne, PA, USA; 2013.
16. Nahavandi K. Calculating Sensitivity, Specificity and Predictive Values for Medical Diagnostic Tests. Gene Cell Tissue. 2018;5(2):e80270.https://doi.org/10.5812/gct.80270
17. Kateete DP, Kimani CN, Katabazi FA, Okeng A, Okee MS, Nanteza A, et al. Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test. Ann Clin Microbiol Antimicrob. 2010;9:23.https://doi.org/10.1186/1476-0711-9-23
18. Aboud WA. Molecular Detection of Vancomycin and Methicillin-Resistant Genes of Staphylococcus aureus Isolated from Animal and Human Specimens. MSc Thesis, College of Veterinary Medicine, University of Basrah; 2019.
19. Mahdi ASH. Molecular Detection of Superantigens in Staphylococcus aureus Isolated from Human, Cattle and Buffalo Milk Samples. MSc Thesis, College of Veterinary Medicine, University of Basrah; 2019.
20. Singh RS, Kumar R, Yadav BR. Distribution of pathogenic factors in Staphylococcus aureus strains isolated from intra-mammary infections in cattle and buffaloes. Indian J Biotechnol. 2011;10:410-6.
21. Turkyilmaz S, Kaya O. Determination of some virulence factors in Staphylococcus spp. isolated from various clinical samples. Turk J Vet Anim Sci. 2005;30:127-32.
22. David FT, Taku A, Bhat MA, Badroo M, Sofi TM. Molecular typing of Staphylococcus aureus based on coagulase gene. Vet World. 2018;11:2231-6.https://doi.org/10.14202/vetworld.2018.423-430
23. Pumipuntu N, Kulpeanprasit S, Santajit S, Tunyong W, Kong-ngoen T, Hinthong W, et al. Screening method for Staphylococcus aureus identification in subclinical bovine mastitis from dairy farms. Vet World. 2017;10:2231-6.https://doi.org/10.14202/vetworld.2017.721-726
24. Gianneechini R, Conch C, Rivero R, Delucci I, Moreno Lopez J. Occurrence of Clinical and Subclinical Mastitis in Dairy Herds in the West Littoral Region in Uruguay. Acta Vet Scand. 2002;43:221-30.https://doi.org/10.1186/1751-0147-43-221
25. Myllys V, Asplund K, Brofeldt E, Hirvela-Koski V, Honkanen-Buzalaske T, Junttila J, et al. Bovine mastitis in Finland in 1988 and 1995-changes in prevalence and antimicrobial resistance. Acta Vet Scand. 1998;39:119-26.https://doi.org/10.1186/BF03547813
26. Pyorala S, Taponen S. Coagulase-negative staphylococci emerging mastitis pathogens. Vet Microbiol. 2009;134:3-8.https://doi.org/10.1016/j.vetmic.2008.09.015
27. AL-Edany A, Khudor M, AL-Mousawi Kh. Comparison of three indirect tests for the diagnosis of bovine subclinical mastitis caused by coagulase-negative staphylococci with their susceptibility to seven antibiotics. Bas J Vet Res. 2012;11(1).https://doi.org/10.33762/bvetr.2012.54759
28. Foster TJ. Immune evasion by staphylococci. Nat Rev Microbiol. 2005;3:948-58.https://doi.org/10.1038/nrmicro1289
29. Palomares C, Torres MJ, Torres A, Aznar J, Palomares JC. Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR. Diagn Microbiol Infect Dis. 2003;45:183-9.https://doi.org/10.1016/S0732-8893(02)00542-4
30. Costa AM, Kay I, Palladino S. Rapid detection of mecA and nuc genes in staphylococci by real-time multiplex polymerase chain reaction. Diagn Microbiol Infect Dis. 2005;51:13-7.https://doi.org/10.1016/j.diagmicrobio.2004.08.014
31. Asfour HAE, Darwish SF. Phenotypic and genotypic detection of both mecA and blaZ genes mediated β-lactam resistance in Staphylococcus strains isolated from bovine mastitis. Glob Vet. 2011;6:39-50.
32. Hammad AM. Microbiological studies on raw milk and some dairy products. MSc Thesis, Minufiya Univ., Egypt; 2004.
33. Rall VLM, Vieira FP, Rall R, Vieitis RL, Fernandes A, Candeias JMG, et al. PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk. Vet Microbiol. 2008;132(3):408-13.https://doi.org/10.1016/j.vetmic.2008.05.011
34. Jahan M, Rahman M, Parvej M, Chowdhury S, Haque M, Talukder M, et al. Isolation and characterization of S. aureus from raw cow milk in Bangladesh. J Adv Vet Anim Res. 2015;2(1):49-59.https://doi.org/10.5455/javar.2015.b47
35. Liu H, Li S, Meng L, Dong L, Zhao S, Lan X, et al. Prevalence, antimicrobial susceptibility, and molecular characterization of S. aureus isolated from dairy herds in northern China. J Dairy Sci. 2017;100(5):340-7.https://doi.org/10.3168/jds.2017-13370
36. Momtaz H, Tajbakhsh E, Rahimi E, Momeni M. Coagulase gene polymorphism of S. aureus isolated from clinical and sub-clinical bovine mastitis in Isfahan and Chaharmahal va Bakhtiari provinces of Iran. Comp Clin Pathol. 2011;20(5):519-22.https://doi.org/10.1007/s00580-010-1029-y
37. Vintov J, Aarestrup FM, Zinn CE, Olsen JE. Association between phage types and antimicrobial resistance among bovine Staphylococcus aureus from 10 countries. Vet Microbiol. 2003;95:133-47.https://doi.org/10.1016/S0378-1135(03)00156-1
38. Idbeis HI. Molecular Analysis of Staphylococcus aureus Isolated From Mastitis and Study Their Biofilm Activity against Cinnamon Oil. PhD thesis, College of Veterinary Medicine, University of Basrah; 2019.
39. Alian F, Rahimi E, Shakerian A, Momtaz H, Riahi M, Momeni M. Antimicrobial resistance of Staphylococcus aureus isolated from bovine, sheep and goat raw milk. Glob Vet. 2012;8:111-4.
40. Al-Iedani AA. Phenotypic study on the capacity of biofilm production in S. aureus isolated from bovine subclinical mastitis and their impact on resistance to antimicrobials. Basrah J Vet Res. 2016;15(2):111-27.https://doi.org/10.33762/bvetr.2016.124293
41. Aliyu Y, Reuben C, Sani A, Salawu E. Occurrence and antibiogram of S. aureus isolated from locally-pasteurised cow milk (kindirmo) sold in parts of Nasarawa Town, Nasarawa State, Nigeria. Microbiol Res J Int. 2018;23(4):1-11.https://doi.org/10.9734/MRJI/2018/40294
42. Michael CA, Domine-Howes D, Labbate M. The antimicrobial resistance crisis: causes, consequences, and management. Front Public Health. 2014;2.https://doi.org/10.3389/fpubh.2014.00145
43. Aldhar CN, Chilgunde SN, Gaikwad RV, Smad A, Roy S. Current trends in diagnosis of mastitis in Veterinary practice. J Remt Vet Corp. 2005;44(3):133-40.
44. Yadav R, Sharma SK, Yadav J, Nathawar P, Kataria AK. Phenotypic and genotypic characterization of Staphylococcus aureus of mastitic milk origin from cattle and buffalo for some virulence properties. J Pure Appl Microbiol. 2009;3(1):353-6.
45. Davis TE, Fuller DD, Aeschleman EC. Rapid, direct identification of Staphylococcus aureus and Streptococcus pneumoniae from blood cultures using commercial immunological kits and modified conventional tests. Diagn Microbiol Infect Dis. 1992;15:295-300.https://doi.org/10.1016/0732-8893(92)90014-K
46. Foster TJ. Immune evasion by staphylococci. Nat Rev Microbiol. 2005;3:948-58.https://doi.org/10.1038/nrmicro1289
47. Palomares C, Torres MJ, Torres A, Aznar J, Palomares JC. Rapid detection and identification of Staphylococcus aureus from blood culture specimens using real-time fluorescence PCR. Diagn Microbiol Infect Dis. 2003;45:183-9.https://doi.org/10.1016/S0732-8893(02)00542-4
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