Evaluation of a field-deployable reverse transcription-insulated isothermal PCR for rapid and sensitive on-site detection of Zika virus

BMC Infectious Diseases

Volumn 17, Issue 1, 2017, Pages

  Carossino, Mariano ^a   Li, Yanqiu ^a   Lee, Pei Yu A ^b   Tsai, Chuan Fu ^b   Chou, Pin Hsing ^b   Williams, Dennis ^c   Skillman, Ashley ^a   Frank Cook, R ^a   Brown, Grayson ^a   Chang, Hsiao Fen G ^b   Wang, Hwa Tang T ^b   Balasuriya, Udeni B R ^a  

^a UNIVERSITY OF KENTUCKY   (United States)

^b GeneReach USA   (United States)

^c UNIVERSITY OF KENTUCKY MEDICAL CENTER   (United States)

Author keywords

IiPCR; Insulated isothermal PCR; POCKIT; Point of need assay; Zika virus

Indexed keywords

RNA; VIRUS RNA;

ANIMAL CELL; ANIMAL TISSUE; ARTICLE; BLOOD ANALYSIS; CONTROLLED STUDY; DIAGNOSTIC TEST ACCURACY STUDY; E GENE; GENE TARGETING; HEALTH CARE SYSTEM; HUMAN; IN VITRO STUDY; INTERMETHOD COMPARISON; MALE; MOSQUITO; NONHUMAN; NS2B GENE; PUBLIC HEALTH SERVICE; REAL TIME POLYMERASE CHAIN REACTION; REVERSE TRANSCRIPTION INSULATED ISOTHERMAL POLYMERASE CHAIN REACTION; REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION; SEMEN ANALYSIS; SENSITIVITY AND SPECIFICITY; URINALYSIS; VIRUS DETECTION; VIRUS GENE; VIRUS STRAIN; WORLD HEALTH ORGANIZATION; ZIKA FEVER; ZIKA VIRUS; ANIMAL; BLOOD; EVALUATION STUDY; GENETICS; ISOLATION AND PURIFICATION; POINT OF CARE SYSTEM; URINE; VIROLOGY;

ANIMALS; CULICIDAE; HUMANS; POINT-OF-CARE SYSTEMS; REAL-TIME POLYMERASE CHAIN REACTION; RNA, VIRAL; SENSITIVITY AND SPECIFICITY; ZIKA VIRUS; ZIKA VIRUS INFECTION;

EID: 85038358642     PISSN: None     EISSN: 14712334     Source Type: Journal    
DOI: 10.1186/s12879-017-2852-4     Document Type: Article

References (86)

1. Dick GW, Kitchen SF, Haddow AJ. Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg. 1952;46(5):509-20.
2. Haddow AJ, Williams MC, Woodall JP, Simpson DI, Goma LK. Twelve isolations of Zika virus from Aedes (Stegomyia) Africanus (Theobald) taken in and above a Uganda Forest. Bull World Health Organ. 1964;31:57-69.
3. Macnamara FN. Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria. Trans R Soc Trop Med Hyg. 1954;48(2):139-45.
4. Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M, Marfel M, Holzbauer S, Dubray C, et al. Zika virus outbreak on Yap Island, Federated States of Micronesia. N Engl J Med. 2009;360(24):2536-43.
5. Plourde AR, Bloch EM. A literature review of Zika virus. Emerg Infect Dis. 2016;22(7):1185-92.
6. Petersen LR, Jamieson DJ, Honein MA. Zika Virus. N Engl J Med. 2016;375(3):294-5.
7. Hayes EB. Zika virus outside Africa. Emerg Infect Dis. 2009;15(9):1347-50.
8. Hennessey M, Fischer M, Staples JE. Zika virus spreads to new areas - region of the Americas, may 2015-January 2016. MMWR Morb Mortal Wkly Rep. 2016;65(3):55-8.
9. Zika virus outbreaks in the Americas. Wkly Epidemiol Rec 2015;90(45):609-10. (PMID 26552108).
10. Campos GS, Bandeira AC, Sardi SI. Zika Virus Outbreak, Bahia. Brazil Emerg Infect Dis. 2015;21(10):1885-6.
11. Zanluca C, Melo VC, Mosimann AL, Santos GI, Santos CN, Luz K. First report of autochthonous transmission of Zika virus in Brazil. Mem Inst Oswaldo Cruz. 2015;110(4):569-72.
12. Thomas DL, Sharp TM, Torres J, Armstrong PA, Munoz-Jordan J, Ryff KR, Martinez-Quinones A, Arias-Berrios J, Mayshack M, Garayalde GJ, et al. Local transmission of Zika virus--Puerto Rico, November 23, 2015-January 28, 2016. MMWR Morb Mortal Wkly Rep. 2016;65(6):154-8.
13. Likos A, Griffin I, Bingham AM, Stanek D, Fischer M, White S, Hamilton J, Eisenstein L, Atrubin D, Mulay P, et al. Local mosquito-borne transmission of Zika virus - Miami-Dade and Broward counties, Florida, June-august 2016. MMWR Morb Mortal Wkly Rep. 2016;65(38):1032-8.
14. Kuno G, Chang GJ, Tsuchiya KR, Karabatsos N, Cropp CB. Phylogeny of the genus Flavivirus. J Virol. 1998;72(1):73-83.
15. Lindenbach BD, Rice CM. Molecular biology of flaviviruses. Adv Virus Res. 2003;59:23-61.
16. Chambers TJ, Hahn CS, Galler R, Rice CM. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. 1990;44:649-88.
17. Lanciotti RS, Kosoy OL, Laven JJ, Velez JO, Lambert AJ, Johnson AJ, Stanfield SM, Duffy MR. Genetic and serologic properties of Zika virus associated with an epidemic, yap state, Micronesia, 2007. Emerg Infect Dis. 2008;14(8):1232-9.
18. Shan C, Xie X, Muruato AE, Rossi SL, Roundy CM, Azar SR, Yang Y, Tesh RB, Bourne N, Barrett AD, et al. An infectious cDNA clone of Zika virus to study viral virulence, mosquito transmission, and antiviral inhibitors. Cell Host Microbe. 2016;19(6):891-900.
19. Lanciotti RS, Lambert AJ, Holodniy M, Saavedra S, Signor Ldel C. Phylogeny of Zika virus in western hemisphere, 2015. Emerg Infect Dis. 2016;22(5):933-5.
20. Ye Q, Liu ZY, Han JF, Jiang T, Li XF, Qin CF. Genomic characterization and phylogenetic analysis of Zika virus circulating in the Americas. Infect Genet Evol. 2016;43:43-9.
21. Thangamani S, Huang J, Hart CE, Guzman H, Tesh RB. Vertical transmission of Zika virus in Aedes Aegypti mosquitoes. Am J Trop Med Hyg. 2016;95(5):1169-73.
22. Marchette NJ, Garcia R, Rudnick A. Isolation of Zika virus from Aedes Aegypti mosquitoes in Malaysia. Am J Trop Med Hyg. 1969;18(3):411-5.
23. Ferreira-de-Brito A, Ribeiro IP, Miranda RM, Fernandes RS, Campos SS, Silva KA, Castro MG, Bonaldo MC, Brasil P, Lourenco-de-Oliveira R. First detection of natural infection of Aedes Aegypti with Zika virus in Brazil and throughout South America. Mem Inst Oswaldo Cruz. 2016;111(10):655-8.
24. Wong PS, Li MZ, Chong CS, Ng LC, Tan CH. Aedes (Stegomyia) albopictus (Skuse): a potential vector of Zika virus in Singapore. PLoS Negl Trop Dis. 2013;7(8):e2348.
25. Grard G, Caron M, Mombo IM, Nkoghe D, Mboui Ondo S, Jiolle D, Fontenille D, Paupy C, Leroy EM. Zika virus in Gabon (Central Africa)--2007: a new threat from Aedes Albopictus? PLoS Negl Trop Dis. 2014;8(2):e2681.
26. Besnard M, Lastere S, Teissier A, Cao-Lormeau V, Musso D. Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014. Euro Surveill. 2014;19(13)
27. D'Ortenzio E, Matheron S, Yazdanpanah Y, de Lamballerie X, Hubert B, Piorkowski G, Maquart M, Descamps D, Damond F, Leparc-Goffart I. Evidence of sexual transmission of Zika virus. N Engl J Med. 2016;374(22):2195-8.
28. Hills SL, Russell K, Hennessey M, Williams C, Oster AM, Fischer M, Mead P. Transmission of Zika virus through sexual contact with travelers to areas of ongoing transmission - continental United States, 2016. MMWR Morb Mortal Wkly Rep. 2016;65:215-6.
29. McCarthy M. US health officials investigate sexually transmitted Zika virus infections. BMJ. 2016;352:i1180.
30. McCarthy M. Zika virus was transmitted by sexual contact in Texas, health officials report. BMJ. 2016;352:i720.
31. Musso D, Roche C, Robin E, Nhan T, Teissier A, Cao-Lormeau VM. Potential sexual transmission of Zika virus. Emerg Infect Dis. 2015;21(2):359-61.
32. Aubry M, Richard V, Green J, Broult J, Musso D. Inactivation of Zika virus in plasma with amotosalen and ultraviolet a illumination. Transfusion. 2016;56(1):33-40.
33. Jimenez A, Shaz BH, Bloch EM. Zika virus and the blood supply: what do we know? Transfus Med Rev. 2017;31(1):1-10.
34. Kuehnert MJ, Basavaraju SV, Moseley RR, Pate LL, Galel SA, Williamson PC, Busch MP, Alsina JO, Climent-Peris C, Marks PW, et al. Screening of blood donations for Zika virus infection - Puerto Rico, April 3-June 11, 2016. MMWR Morb Mortal Wkly Rep. 2016;65(24):627-8.
35. Galel SA, Williamson PC, Busch MP, Stanek D, Bakkour S, Stone M, Lu K, Jones S, Rossmann SN, Pate LL, et al. First Zika-positive donations in the continental United States. Transfusion. 2017; https://doi.org/10.1111/trf.14029.
36. Barjas-Castro ML, Angerami RN, Cunha MS, Suzuki A, Nogueira JS, Rocco IM, Maeda AY, Vasami FG, Katz G, Boin IF, et al. Probable transfusion-transmitted Zika virus in Brazil. Transfusion. 2016;56(7):1684-8.
37. Driggers RW, Ho CY, Korhonen EM, Kuivanen S, Jaaskelainen AJ, Smura T, Rosenberg A, Hill DA, DeBiasi RL, Vezina G, et al. Zika virus infection with prolonged maternal Viremia and fetal brain abnormalities. N Engl J Med. 2016;374(22):2142-51.
38. Mlakar J, Korva M, Tul N, Popovic M, Poljsak-Prijatelj M, Mraz J, Kolenc M, Resman Rus K, Vesnaver Vipotnik T, Fabjan Vodusek V, et al. Zika virus associated with Microcephaly. N Engl J Med. 2016;374(10):951-8.
39. Tetro JA. Zika and microcephaly: causation, correlation, or coincidence? Microbes Infect. 2016;18(3):167-8.
40. Schuler-Faccini L, Ribeiro EM, Feitosa IM, Horovitz DD, Cavalcanti DP, Pessoa A, Doriqui MJ, Neri JI, Neto JM, Wanderley HY, et al. Possible association between Zika virus infection and Microcephaly - Brazil, 2015. MMWR Morb Mortal Wkly Rep. 2016;65(3):59-62.
41. Acevedo N, Waggoner J, Rodriguez M, Rivera L, Landivar J, Pinsky B, Zambrano H. Zika virus, Chikungunya virus, and dengue virus in cerebrospinal fluid from adults with neurological manifestations, Guayaquil, Ecuador. Front Microbiol. 2017;8:42.
42. Krauer F, Riesen M, Reveiz L, Oladapo OT, Martinez-Vega R, Porgo TV, Haefliger A, Broutet NJ, Low N, Group WHOZCW. Zika virus infection as a cause of congenital brain abnormalities and Guillain-Barre syndrome: systematic review. PLoS Med. 2017;14(1):e1002203.
43. Waggoner JJ, Pinsky BA. Zika virus: diagnostics for an emerging pandemic threat. J Clin Microbiol. 2016;54(4):860-7.
44. Charrel RN, Leparc-Goffart I, Pas S, de Lamballerie X, Koopmans M, Reusken C. Background review for diagnostic test development for Zika virus infection. Bull World Health Organ. 2016;94(8):574-584D.
45. Landry ML, St George K. Laboratory diagnosis of Zika virus infection. Arch Pathol Lab Med. 2017;141(1):60-7.
46. Faye O, Faye O, Diallo D, Diallo M, Weidmann M, Sall AA. Quantitative real-time PCR detection of Zika virus and evaluation with field-caught mosquitoes. Virol J. 2013;10:311-2.
47. Faye O, Faye O, Dupressoir A, Weidmann M, Ndiaye M, Alpha Sall A. One-step RT-PCR for detection of Zika virus. J Clin Virol. 2008;43(1):96-101.
48. Waggoner JJ, Gresh L, Mohamed-Hadley A, Ballesteros G, Davila MJ, Tellez Y, Sahoo MK, Balmaseda A, Harris E, Pinsky BA. Single-reaction multiplex reverse transcription PCR for detection of Zika, Chikungunya, and dengue viruses. Emerg Infect Dis. 2016;22(7):1295-7.
49. Balm MN, Lee CK, Lee HK, Chiu L, Koay ES, Tang JW. A diagnostic polymerase chain reaction assay for Zika virus. J Med Virol. 2012;84(9):1501-5.
50. Corman VM, Rasche A, Baronti C, Aldabbagh S, Cadar D, Reusken CB, Pas SD, Goorhuis A, Schinkel J, Molenkamp R, et al. Assay optimization for molecular detection of Zika virus. Bull World Health Organ. 2016;94(12):880-92.
51. Xu MY, Liu SQ, Deng CL, Zhang QY, Zhang B. Detection of Zika virus by SYBR green one-step real-time RT-PCR. J Virol Methods. 2016;236:93-7.
52. Pardee K, Green AA, Takahashi MK, Braff D, Lambert G, Lee JW, Ferrante T, Ma D, Donghia N, Fan M, et al. Rapid, low-cost detection of Zika virus using programmable biomolecular components. Cell. 2016;165(5):1255-66.
53. Eboigbodin KE, Brummer M, Ojalehto T, Hoser M. Rapid molecular diagnostic test for Zika virus with low demands on sample preparation and instrumentation. Diagn Microbiol Infect Dis. 2016;86(4):369-71.
54. Song J, Mauk MG, Hackett BA, Cherry S, Bau HH, Liu C. Instrument-free point-of-care molecular detection of Zika virus. Anal Chem. 2016;88(14):7289-94.
55. Chang HF, Tsai YL, Tsai CF, Lin CK, Lee PY, Teng PH, Su C, Jeng CC. A thermally baffled device for highly stabilized convective PCR. Biotechnol J. 2012;7(5):662-6.
56. Go YY, Rajapakse RP, Kularatne SA, Lee PY, Ku KB, Nam S, Chou PH, Tsai YL, Liu YL, Chang HF, et al. A pan-dengue virus reverse transcription-insulated isothermal PCR assay intended for point-of-need diagnosis of dengue virus infection by use of the POCKIT nucleic acid analyzer. J Clin Microbiol. 2016;54(6):1528-35.
57. Chua KH, Lee PC, Chai HC. Development of insulated isothermal PCR for rapid on-site malaria detection. Malar J. 2016;15:134-7.
58. Go YY, Kim YS, Cheon S, Ku KB, Nam SW, Kim M, Lee P-YA, Lin Y-C, Tsai Y-L, 1146 Wang H-TT et al: Evaluation and clinical validation of a field-deployable Reverse Transcription-Insulated Isothermal PCR Assay for the Detection of the Middle East Respiratory Syndrome Coronavirus. J Mol Diagn 2017;19(6):817-827.
59. Tsai YL, Wang HC, Lo CF, Tang-Nelson K, Lightner D, Ou BR, Hour AL, Tsai CF, Yen CC, Chang HF, et al. Validation of a commercial insulated isothermal PCR-based POCKIT test for rapid and easy detection of white spot syndrome virus infection in Litopenaeus Vannamei. PLoS One. 2014;9(3):e90545.
60. Balasuriya UB, Lee PA, Tsai YL, Tsai CF, Shen YH, Chang HG, Skillman A, Wang HT, Pronost S, Zhang Y. Translation of a laboratory-validated equine herpesvirus-1 specific real-time PCR assay into an insulated isothermal polymerase chain reaction (iiPCR) assay for point-of-need diagnosis using POCKIT nucleic acid analyzer. J Virol Methods. 2017;241:58-63.
61. Wilkes RP, Kania SA, Tsai YL, Lee PY, Chang HH, Ma LJ, Chang HF, Wang HT. Rapid and sensitive detection of feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform. J Vet Diagn Investig. 2015;27(4):510-5.
62. Wilkes RP, Tsai YL, Lee PY, Lee FC, Chang HF, Wang HT. Rapid and sensitive detection of canine distemper virus by one-tube reverse transcription-insulated isothermal polymerase chain reaction. BMC Vet Res. 2014;10:213.
63. Wilkes RP, Lee PY, Tsai YL, Tsai CF, Chang HH, Chang HF, Wang HT. An insulated isothermal PCR method on a field-deployable device for rapid and sensitive detection of canine parvovirus type 2 at points of need. J Virol Methods. 2015;220:35-8.
64. Zhang J, Tsai YL, Lee PY, Chen Q, Zhang Y, Chiang CJ, Shen YH, Li FC, Chang HF, Gauger PC, et al. Evaluation of two singleplex reverse transcription-insulated isothermal PCR tests and a duplex real-time RT-PCR test for the detection of porcine epidemic diarrhea virus and porcine deltacoronavirus. J Virol Methods. 2016;234:34-42.
65. Tsai YL, Wang HT, Chang HF, Tsai CF, Lin CK, Teng PH, Su C, Jeng CC, Lee PY. Development of TaqMan probe-based insulated isothermal PCR (iiPCR) for sensitive and specific on-site pathogen detection. PLoS One. 2012;7(9):e45278.
66. Carossino M, Lee PY, Nam B, Skillman A, Shuck KM, Timoney PJ, Tsai YL, Ma LJ, Chang HF, Wang HT, et al. Development and evaluation of a reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay for detection of equine arteritis virus in equine semen and tissue samples using the POCKIT system. J Virol Methods. 2016;234:7-15.
67. Tsai YL, Lin YC, Chou PH, Teng PH, Lee PY. Detection of white spot syndrome virus by polymerase chain reaction performed under insulated isothermal conditions. J Virol Methods. 2012;181(1):134-7.
68. Soltan MA, Tsai YL, Lee PY, Tsai CF, Chang HF, Wang HT, Wilkes RP. Comparison of electron microscopy, ELISA, real time RT-PCR and insulated isothermal RT-PCR for the detection of rotavirus group a (RVA) in feces of different animal species. J Virol Methods. 2016;235:99-104.
69. Ambagala A, Pahari S, Fisher M, Lee PA, Pasick J, Ostlund EN, Johnson DJ, Lung O. A rapid field-deployable reverse transcription-insulated isothermal polymerase chain reaction assay for sensitive and specific detection of bluetongue virus. Transbound Emerg Dis. 2015; https://doi.org/10.1111/tbed.12554.
70. Ambagala A, Fisher M, Goolia M, Nfon C, Furukawa-Stoffer T, Ortega Polo R, Lung O. Field-deployable reverse transcription-insulated isothermal PCR (RT-iiPCR) assay for rapid and sensitive detection of foot-and-mouth disease virus. Transbound Emerg Dis. 2016; https://doi.org/10.1111/tbed.12554.
71. WHO statement on the first meeting of the International Health Regulations (2005) (IHR 2005) Emergency Committee on Zika virus and observed increase in neurological disorders and neonatal malformations. http://www.who.int/mediacentre/news/statements/2016/1st-emergency-committee-zika/en/. In.: World Health Organization; 1 February 2016.
72. Froeschl G, Huber K, von Sonnenburg F, Nothdurft HD, Bretzel G, Hoelscher M, Zoeller L, Trottmann M, Pan-Montojo F, Dobler G, et al. Long-term kinetics of Zika virus RNA and antibodies in body fluids of a vasectomized traveller returning from Martinique: a case report. BMC Infect Dis. 2017;17(1):55.
73. Mansuy JM, Dutertre M, Mengelle C, Fourcade C, Marchou B, Delobel P, Izopet J, Martin-Blondel G. Zika virus: high infectious viral load in semen, a new sexually transmitted pathogen? Lancet Infect Dis. 2016;16(4):405.
74. Mansuy JM, Suberbielle E, Chapuy-Regaud S, Mengelle C, Bujan L, Marchou B, Delobel P, Gonzalez-Dunia D, Malnou CE, Izopet J, et al. Zika virus in semen and spermatozoa. Lancet Infect Dis. 2016;16(10):1106-7.
75. Matheron S, d'Ortenzio E, Leparc-Goffart I, Hubert B, de Lamballerie X, Yazdanpanah Y. Long-lasting persistence of Zika virus in semen. Clin Infect Dis. 2016;63(9):1264.
76. Nicastri E, Castilletti C, Liuzzi G, Iannetta M, Capobianchi MR, Ippolito G. Persistent detection of Zika virus RNA in semen for six months after symptom onset in a traveller returning from Haiti to Italy, February 2016. Euro Surveill. 2016;21(32) https://doi.org/10.2807/1560-7917.ES.2016.2821.2832.30314.
77. Musso D, Nhan T, Robin E, Roche C, Bierlaire D, Zisou K, Shan Yan A, Cao-Lormeau VM, Broult J. Potential for Zika virus transmission through blood transfusion demonstrated during an outbreak in French Polynesia, November 2013 to February 2014. Euro Surveill. 2014;19(14)
78. Zambrano H, Waggoner J, Leon K, Pinsky B, Vera K, Schettino M, Rivera L, Landivar J, Granda M, Lee A et al: High incidence of Zika virus infection detected in plasma and cervical cytology specimens from pregnant women in Guayaquil, Ecuador. Am J Reprod Immunol 2017, 77(2):doi: https://doi.org/10.1111/aji.12630.
79. Paz-Bailey G, Rosenberg ES, Doyle K, Munoz-Jordan J, Santiago GA, Klein L, Perez-Padilla J, Medina FA, Waterman SH, Gubern CG, et al. Persistence of Zika virus in body fluids - preliminary report. N Engl J Med. 2017; https://doi.org/10.1056/NEJMoa1613108.
80. Murray KO, Gorchakov R, Carlson AR, Berry R, Lai L, Natrajan M, Garcia MN, Correa A, Patel SM, Aagaard K, et al. Prolonged detection of Zika virus in vaginal secretions and whole blood. Emerg Infect Dis. 2017;23(1):99-101.
81. Barzon L, Pacenti M, Franchin E, Lavezzo E, Trevisan M, Sgarabotto D, Palu G. Infection dynamics in a traveller with persistent shedding of Zika virus RNA in semen for six months after returning from Haiti to Italy, January 2016. Euro Surveill. 2016;21(32). doi: 10.2807/1560-7917.ES.2016.21.32.30316.
82. Bonaldo MC, Ribeiro IP, Lima NS, Dos Santos AA, Menezes LS, da Cruz SO, de Mello IS, Furtado ND, de Moura EE, Damasceno L, et al. Isolation of infective Zika virus from urine and saliva of patients in Brazil. PLoS Negl Trop Dis. 2016;10(6):e0004816.
83. Abd El Wahed A, Sanabani SS, Faye O, Pessoa R, Patriota JV, Giorgi RR, Patel P, Bohlken-Fascher S, Landt O, Niedrig M, et al. Rapid molecular detection of Zika virus in acute-phase urine samples using the Recombinase polymerase amplification assay. PLOS Currents Outbreaks. 2017; https://doi.org/10.1371/currents.outbreaks.a1377f1371db1372c1377d1366c1373fc1370ea1370a774305d774319e.
84. Bustin SA, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech. 2004;15(3):155-66.
85. Fleige S, Pfaffl MW. RNA integrity and the effect on the real-time qRT-PCR performance. Mol Asp Med. 2006;27(2-3):126-39.
86. Schrader C, Schielke A, Ellerbroek L, Johne R. PCR inhibitors - occurrence, properties and removal. J Appl Microbiol. 2012;113(5):1014-26.