Development and validation of rapid magnetic particle based extraction protocols

Virology Journal

Bernd Hoffmann¹  Martin Beer¹  Andrea Aebischer¹ 

[1] Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Suedufer 10, 17493 Greifwald-Insel Riems, Germany

关键词: Mobile diagnostic laboratory;    Bovine viral diarrhea virus;    Schmallenberg virus;    Magnetic particle;    Nucleic acid extraction;   

Others  :  1148764
DOI  :  10.1186/1743-422X-11-137

 received in 2014-04-30, accepted in 2014-07-27,  发布年份 2014

【 摘 要 】

Background

In order to control and eradicate transboundary animal diseases, early diagnosis and reaction is essential for the implementation of control activities. Thus, mobile diagnostic units which allow analytical testing close to the site of occurrence could provide valuable support for centralized laboratories. Consequently, the availability of diagnostic tests using mobile amplification and detection technologies has been increasing over the past years. However, methods enabling rapid and simple nucleic acid extraction also under resource-limited settings are still scarce.

Methods

In the present study rapid extraction protocols based on magnetic particle technology have been developed. For this purpose, the two open extraction platforms KingFisher™ Duo (Thermo Fisher Scientific) and BioSprint® 15 (Qiagen) as well as the fully automated EZ1® advanced XL instrument (Qiagen) were used. All protocols were validated in comparison to standard manual extraction using blood and serum samples from animals infected with Schmallenberg virus or bovine viral diarrhea virus.

Results

All newly developed protocols allowed a complete extraction within 30 minutes of time. The fully automated EZ1-extraction yielded the highest reproducibility, whereas slightly higher intra- and inter-assay variations were observed using the open platforms. Compared to the manual procedure, the analytical sensitivity of all the rapid protocols was 1 log₁₀ step reduced for extraction from blood samples. For sera a reduced dynamic range could only be observed using the maximally shortened BioSprint 15 protocol. Validation using clinical samples showed an excellent concordance of all the rapid extraction protocols to the standard manual extraction procedure, independent of sample materials and target viruses.

Conclusions

The results of this study show that the speed-optimized novel extraction protocols allow rapid and simple nucleic acid extractions for a variety of target viruses without significant loss of sensitivity compared to standard procedures. For this reason they represent valuable tools to accelerate magnetic particle based automated extraction.

【 授权许可】

   
2014 Aebischer et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Niemz A, Ferguson TM, Boyle DS: Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol 2011, 29:240-250.
• [2]Holland CA, Kiechle FL: Point-of-care molecular diagnostic systems–past, present and future. Curr Opin Microbiol 2005, 8:504-509.
• [3]Park S, Zhang Y, Lin S, Wang TH, Yang S: Advances in microfluidic PCR for point-of-care infectious disease diagnostics. Biotechnol Adv 2011, 29:830-839.
• [4]Craw P, Balachandran W: Isothermal nucleic acid amplification technologies for point-of-care diagnostics: a critical review. Lab Chip 2012, 12:2469-2486.
• [5]Asiello PJ, Baeumner AJ: Miniaturized isothermal nucleic acid amplification, a review. Lab Chip 2011, 11:1420-1430.
• [6]Raja S, Ching J, Xi L, Hughes SJ, Chang R, Wong W, McMillan W, Gooding WE, McCarty KS Jr, Chestney M, Luketich JD, Godfrey TE: Technology for automated, rapid, and quantitative PCR or reverse transcription-PCR clinical testing. Clin Chem 2005, 51:882-890.
• [7]GeneXpert, Cepheidhttp://www.cepheid.com/en/cepheid-solutions-uk/systems/genexpert-systems/genexpert-i webcite
• [8]Tanriverdi S, Chen L, Chen S: A rapid and automated sample-to-result HIV load test for near-patient application. J Infect Dis 2010, 201(Suppl 1):S52-S58.
• [9]Liat Analyzer, IQuumhttp://www.iquum.com/products/analyzer.shtml webcite
• [10]Enigma ML, Enigma Diagnosticshttp://www.enigmadiagnostics.com/template2.php?page=instruments.php webcite
• [11]Fuehrer HP, Fally MA, Habler VE, Starzengruber P, Swoboda P, Noedl H: Novel nested direct PCR technique for malaria diagnosis using filter paper samples. J Clin Microbiol 2011, 49:1628-1630.
• [12]Mens PF, de Bes HM, Sondo P, Laochan N, Keereecharoen L, van Amerongen A, Flint J, Sak JR, Proux S, Tinto H, Schallig HD: Direct blood PCR in combination with nucleic acid lateral flow immunoassay for detection of Plasmodium species in settings where malaria is endemic. J Clin Microbiol 2012, 50:3520-3525.
• [13]Verheij S, Harteveld J, Sijen T: A protocol for direct and rapid multiplex PCR amplification on forensically relevant samples. Forensic Sci Int Genet 2012, 6:167-175.
• [14]Dineva MA, MahiLum-Tapay L, Lee H: Sample preparation: a challenge in the development of point-of-care nucleic acid-based assays for resource-limited settings. Analyst 2007, 132:1193-1199.
• [15]Hoffmann B, Scheuch M, Hoper D, Jungblut R, Holsteg M, Schirrmeier H, Eschbaumer M, Goller KV, Wernike K, Fischer M, Breithaupt A, Mettenleiter TC, Beer M: Novel orthobunyavirus in Cattle, Europe, 2011. Emerg Infect Dis 2012, 18:469-472.
• [16]Beer M, Conraths FJ, van der Poel WH: ‘Schmallenberg virus’–a novel orthobunyavirus emerging in Europe. Epidemiol Infect 2013, 141:1-8.
• [17]Wernike K, Conraths F, Zanella G, Granzow H, Gache K, Schirrmeier H, Valas S, Staubach C, Marianneau P, Kraatz F, Höreth-Böntgen D, Reimann I, Zientara S, Beer M: Schmallenberg virus-Two years of experiences. Prev Vet Med 2014. doi:10.1016/j.prevetmed.2014.03.021
• [18]Doceul V, Lara E, Sailleau C, Belbis G, Richardson J, Breard E, Viarouge C, Dominguez M, Hendrikx P, Calavas D, Desprat A, Languille J, Comtet L, Pourquier P, Eleouet JF, Delmas B, Marianneau P, Vitour D, Zientara S: Epidemiology, molecular virology and diagnostics of Schmallenberg virus, an emerging orthobunyavirus in Europe. Vet Res 2013, 44:31.
• [19]Neill JD: Molecular biology of bovine viral diarrhea virus. Biologicals 2013, 41:2-7.
• [20]Presi P, Struchen R, Knight-Jones T, Scholl S, Heim D: Bovine viral diarrhea (BVD) eradication in Switzerland–experiences of the first two years. Prev Vet Med 2011, 99:112-121.
• [21]Stahl K, Alenius S: BVDV control and eradication in Europe–an update. Jpn J Vet Res 2012, 60(Suppl):S31-S39.
• [22]Bilk S, Schulze C, Fischer M, Beer M, Hlinak A, Hoffmann B: Organ distribution of Schmallenberg virus RNA in malformed newborns. Vet Microbiol 2012, 159:236-238.
• [23]Hyndman L, Vilcek S, Conner J, Nettleton PF: A novel nested reverse transcription PCR detects bovine viral diarrhoea virus in fluids from aborted bovine fetuses. J Virol Methods 1998, 71:69-76.
• [24]Gaede W, Reiting R, Schirrmeier H, Depner KR, Beer M: Detection and species-specific differentiation of pestiviruses using real-time RT-PCR. Berl Munch Tierarztl Wochenschr 2005, 118:113-120.
• [25]Hoffmann B, Depner K, Schirrmeier H, Beer M: A universal heterologous internal control system for duplex real-time RT-PCR assays used in a detection system for pestiviruses. J Virol Methods 2006, 136:200-209.
• [26]Al-Soud WA, Radstrom P: Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 2001, 39:485-493.
• [27]Radstrom P, Knutsson R, Wolffs P, Lovenklev M, Lofstrom C: Pre-PCR processing: strategies to generate PCR-compatible samples. Mol Biotechnol 2004, 26:133-146.