Evaluation of CMV R-gene PCR (Argene) coupled with Easy ... · Evaluation of CMV R-gene PCR...
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Sophie Alain 1,2 , Béatrice Boyer 1 , Matthieu Vignolles 3 , Martine Joannes 3 , Côme Barranger 3 , Marie cécile Ploy 2 1 : French National reference center for Cytomegaloviruses, CHU Dupuytren, Limoges France 2 : Bacteriology –Virology-Hygiene department, CHU Dupuytren, Limoges, France 3 : Argene Corporation, Varhiles, France sophie.alain@unilim. Evaluation of CMV R-gene PCR (Argene) coupled with Easy Mag Biomérieux extraction for CMV viral load quantification in amniotic fluid Methods Introduction Results Acknowledgements : Argene (France) for providing thePCR assay kits Diagnosis of Cytomegalovirus (CMV) congenital infection in utero relies on viral DNA detection in amniotic fluid. Though many PCR methods have been applied to CMV viral load measurement, only few of them are currently validated for antenatal diagnosis. In particular, extraction methods, which can influence both reproducibility and sensitivity of PCR in such cellular-rich fluids, have to be cautiously analysed. We herein aimed to study the performances of the CMV R-gene PCR kit (Argene, France), CE marked for AF with Qiagen DNA blood and M2000 Abbott extraction methods, and used here with the automated Easy-Mag extraction (BioMérieux, France) method. Materials •Positive samples for comparison of methods and reproducibility after conservation • 2 CMV-negative amniotic fluids spiked with high titer saliva sample (10 e 8 copies/ml) N° 506 and N° 322 • 2 CMV-positive amniotic fluids diluted in negative amniotic fluid N° DEM and N° K. •Positive control: International Standard diluted in negative amniotic fluid from 6,7 to 0,7 log copies/mL • CMV-negative samples for specificity (not diluted) • 6 negative amniotic fluids • 5 positive amniotic fluids (2 for Parvovirus B19, 1 for HSV1, 1 for Enterovirus, 1 for HHV6) All these samples were kept frozen at -80°C before extraction. QuanBtaBve real‐Bme PCR assay In-house UL83 assay: Taqman assay with UDG (Mengelle et al., J. Med Virol, 2003) Light Cycler® 1.0 (Roche) external control with albumin PCR (Wagner et al., J. Virol. Methods, 2007) Technically and in clinical practice validated Used in many laboratories until 2010 Conclusion 10μL P 118 Easy Mag : Sample: 200ul Pre-lysis with PK at 56°C Protocol B Elution 100uL DNA blood Qiagen: Sample: 200ul Manual minicolumn extraction Elution 100uL M2000sp: Sample: 200ul Elution: 100uL CMV Rgene assay: Taqman assay with UDG Rotor gene apparatus Internal control in multiplex CE marked for amniotic fluid with Qiagen DNA blood and M2000 Abbott extraction methods All samples and dilution were extracted and tested in parallel with both PCR combined with each extraction method. Impact of extraction method on CMV quantification 10,00 15,00 20,00 25,00 30,00 35,00 0 1 2 3 4 5 6 7 8 PCR cycles repeats easyMAG/R-gene 1 easyMAG/R-gene 2 Qiagen/R-gene 1 Qiagen/R-gene 2 M2000/R-gene IC variation upon various extractions on amniotic fluid N° DEM 10,00 15,00 20,00 25,00 30,00 35,00 0 2 4 6 8 10 PCR cycles Repeats IC variation upon various extractions on amniotic fluid N°506 Specificity : 100% all the samples were negaBve for CMV with both assays Reproducibility was tested on the internal control : Internal control values were highly reproducible with the Rgene assay CMV‐negaBve samples : mean value 27, 05 +/‐0,46 cycles PosiBve control: 24,06+/‐ 0,18 (Easy Mag bioMérieux) versus 27,54+/‐2,27 cycle (DNA blood Qiagen) PosiBve samples: 28,54+/‐0,82 (AbboX M200sp), 24,48+/‐0,41 ; 25,95+/‐0,38, 26,3+/‐0,26 (Easy Mag bioMérieux), 26,3+/‐1,7; 26,50+/‐0,68; 25,91+/‐0,79 (DNA blood Qiagen) CMV R-gene internal controls were highly reproducible with Easy Mag showing the good performance of the extraction method. On diluted samples and on the standard the four combinations show good correlation and a high linearity from 6 to 2 log without any saturation effect for highest viral loads. The three combinations of CMV Rgene assay with either manualDNA blood, Easy mag or M2000 sp are reliable for CMV load measurement in amniotic fluid, though these results have to be confirmed on a panel of CMV-positive undiluted AF. 0 1 2 3 4 5 6 7 8 pur dilution 1: 5,70 log dilution 2: 4,7 log dilution 3: 3,7 log dilution 4: 2,7 log dilution 5: 1,7 log dilution 6: 0,7 log log ( cop/ml) International Standard EASYMAG + RGENE EASYMAG + UL83 QIAGEN + RGENE QIAGEN + UL83 TARGET VALUE 0 2 4 6 8 10 12 A B C D E F G H I log copies/mL SAMPLES Amniotic fluid N°506 EASY MAG1 + RGENE1 EASYMAG 1+ UL83 QIAGEN1 + RGENE1 QIAGEN1 + UL83 M2000 + RGENE M2000 + UL83 TARGET VALUE EASYMAG 2 + RGENE 2 EASYMAG 3 + RGENE3 QIAGEN 2 + RGENE2 QIAGEN 3 + RGENE 3 0 1 2 3 4 5 6 7 8 A B C D E F G log copies/mL Amniotic fluid N°DEM • Viral load quantification was lower with the UL83 than with Rgene assay whatever the extraction method and the positive sample or standard. • From the International standard we observe an excellent correlation between Easy mag extraction and manual DNA blood extraction with both methods (R2= 0,99 for Rgene and 0,94 for UL83). Easy mag values were slightly higher than Qiagen values for both methods (mean 0,32+/-0,21 for R gene). • The lower viral load samples were consistently detected at 3 log copies/mL in samples and in the standard but not at lower values
Transcript of Evaluation of CMV R-gene PCR (Argene) coupled with Easy ... · Evaluation of CMV R-gene PCR...
Sophie Alain 1,2, Béatrice Boyer 1, Matthieu Vignolles 3, Martine Joannes3, Côme Barranger 3, Marie cécile Ploy 2 1 : French National reference center for Cytomegaloviruses, CHU Dupuytren, Limoges France
2 : Bacteriology –Virology-Hygiene department, CHU Dupuytren, Limoges, France 3 : Argene Corporation, Varhiles, France
sophie.alain@unilim.
Evaluation of CMV R-gene PCR (Argene) coupled with Easy Mag Biomérieux extraction for CMV viral load quantification in amniotic fluid
Methods
Introduction Results
Acknowledgements:Argene(France)forprovidingthePCRassaykits
Diagnosis of Cytomegalovirus (CMV) congenital infection in utero relies on viral DNA detection in amniotic fluid. Though many PCR methods have been applied to CMV viral load measurement, only few of them are currently validated for antenatal diagnosis. In particular, extraction methods, which can influence both reproducibility and sensitivity of PCR in such cellular-rich fluids, have to be cautiously analysed. We herein aimed to study the performances of the CMV R-gene PCR kit (Argene, France), CE marked for AF with Qiagen DNA blood and M2000 Abbott extraction methods, and used here with the automated Easy-Mag extraction (BioMérieux, France) method.
Materials
• Positive samples for comparison of methods and reproducibility after conservation
• 2 CMV-negative amniotic fluids spiked with high titer saliva sample (10e8 copies/ml) N° 506 and N° 322
• 2 CMV-positive amniotic fluids diluted in negative amniotic fluid N° DEM and N° K.
• Positive control: International Standard diluted in negative amniotic fluid from 6,7 to 0,7 log copies/mL
• CMV-negative samples for specificity (not diluted) • 6 negative amniotic fluids • 5 positive amniotic fluids (2 for Parvovirus B19, 1 for HSV1, 1 for
Enterovirus, 1 for HHV6)
All these samples were kept frozen at -80°C before extraction.
QuanBtaBvereal‐BmePCRassay
In-house UL83 assay:
Taqman assay with UDG (Mengelle et al., J. Med Virol, 2003) Light Cycler® 1.0 (Roche) external control with albumin PCR (Wagner et al., J. Virol. Methods, 2007) Technically and in clinical practice validated Used in many laboratories until 2010
Conclusion
10µL
P 118
Easy Mag : Sample: 200ul Pre-lysis with PK at 56°C Protocol B Elution 100uL
DNA blood Qiagen: Sample: 200ul Manual minicolumn extraction Elution 100uL
M2000sp: Sample: 200ul Elution: 100uL
CMV Rgene assay:
Taqman assay with UDG Rotor gene apparatus Internal control in multiplex CE marked for amniotic fluid with Qiagen DNA blood and M2000 Abbott extraction methods
All samples and dilution were extracted and tested in parallel with both PCR combined with each extraction method.
Impact of extraction method on CMV quantification
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15,00
20,00
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35,00
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PCR c
ycle
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easyMAG/R-gene 1
easyMAG/R-gene 2
Qiagen/R-gene 1
Qiagen/R-gene 2
M2000/R-gene
IC variation upon various extractions on amniotic fluid N° DEM
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35,00
0 2 4 6 8 10
PCR c
ycle
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Repeats
IC variation upon various extractions on amniotic fluid N°506
Specificity : 100% allthesampleswerenegaBveforCMVwithbothassays
Reproducibility was tested on the internal control : InternalcontrolvalueswerehighlyreproduciblewiththeRgeneassayCMV‐negaBvesamples:meanvalue 27,05+/‐0,46cyclesPosiBvecontrol: 24,06+/‐0,18(EasyMagbioMérieux)versus27,54+/‐2,27cycle(DNAbloodQiagen)PosiBvesamples: 28,54+/‐0,82(AbboXM200sp),24,48+/‐0,41;25,95+/‐0,38,26,3+/‐0,26(Easy MagbioMérieux),26,3+/‐1,7;26,50+/‐0,68;25,91+/‐0,79(DNAbloodQiagen)
CMV R-gene internal controls were highly reproducible with Easy Mag showing the good performance of the extraction method. On diluted samples and on the standard the four combinations show good correlation and a high linearity from 6 to 2 log without any saturation effect for highest viral loads. The three combinations of CMV Rgene assay with either manualDNA blood, Easy mag or M2000 sp are reliable for CMV load measurement in amniotic fluid, though these results have to be confirmed on a panel of CMV-positive undiluted AF.
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pur dilution 1: 5,70 log
dilution 2: 4,7 log
dilution 3: 3,7 log
dilution 4: 2,7 log
dilution 5: 1,7 log
dilution 6: 0,7 log
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International Standard
EASYMAG + RGENE
EASYMAG + UL83
QIAGEN + RGENE
QIAGEN + UL83
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EASY MAG1 + RGENE1 EASYMAG 1+ UL83 QIAGEN1 + RGENE1 QIAGEN1 + UL83 M2000 + RGENE M2000 + UL83 TARGET VALUE EASYMAG 2 + RGENE 2 EASYMAG 3 + RGENE3 QIAGEN 2 + RGENE2 QIAGEN 3 + RGENE 3 0
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Amniotic fluid N°DEM
• Viral load quantification was lower with the UL83 than with Rgene assay whatever the extraction method and the positive sample or standard.
• From the International standard we observe an excellent correlation between Easy mag extraction and manual DNA blood extraction with both methods (R2= 0,99 for Rgene and 0,94 for UL83). Easy mag values were slightly higher than Qiagen values for both methods (mean 0,32+/-0,21 for R gene).
• The lower viral load samples were consistently detected at 3 log copies/mL in samples and in the standard but not at lower values
