MagNA Pure System Application Note No. 1

Abstract

In our diagnostic laboratory, we routinely use theMagNA Pure 96 System for nucleic acid extraction. In thispaper, we show that the MagNA Pure 96 System is reliableand convenient for the extraction of viral RNA and/or DNAfrom a variety of sample materials.

Extraction was very sensitive and effi cient, resulting in the detection of 10 – 100 viral copies, depending on sample and virus type. Effects resulting from PCR inhibition wereexcluded by experiments using Internal Controls. Furthermore, we evaluated one single extraction protocol and one single RT-PCR protocol that can be used genericallyfor all sample and virus types in the same run.

Detection using the Roche LightCycler ® 480 Real-TimePCR Instrument revealed that extraction from all 6 materials tested was very efficient. Even with known difficult materials, such as faeces and biopsies, the MagNA Pure 96 System performed well.

In addition, we extracted nucleic acid from 2 differentvolume inputs (50 and 200 μl) of EDTA whole blood frompatients positive for CMV. Higher inputs led to highersensitivity in 7/8 samples, crossing point (Cp) cycle valueswere approximately 1.7 cycle lower while Cp values for ICremained similar.

René Minnaar and Richard Molenkamp

Department of Medical Microbiology,

Academic Medical Center, (AMC),

Amsterdam, The Netherlands

March 2015

Performance Efficient Extractionof Viral DNA and RNA from aVariety of Human SampleMaterials using a Single Standardized Protocol with the MagNA Pure 96 System

Introduction

The Academic Medical Center (AMC) is one of the eightuniversity medical centers in the Netherlands. The threeprincipal tasks are patient care, research and medicaleducation. The AMC has over 1,000 beds and more than50,000 clinical and daycare admissions. The Departmentof Medical Microbiology participates in all three principaltasks of the AMC. Patient care is provided by laboratorydiagnosis and consultation. Within the department ofmedical microbiology, the molecular diagnostic unit (MDU)provides molecular detection of a broad range ofmicroorganisms. Over the last decade, the contribution of molecular techniques to the diagnosis of infectious disease in medicalmicrobiology laboratories has increased significantly. Not only has the amount of samples pro-cessed and the choice of targets increased, the nature and variety of the sample materials to be tested have grown.

Material Pretreatment Viral Target Sample Volume** Elution Volume Isolation Protocol

EDTA whole blood none CMV, HSV, VZV, EBV 50 μl 50 μl total NA external lysis

Plasma none BK, Parvo, HHV8 200 μl 50 μl total NA external lysis

CSF none EV, HPeV, HSV, JC, VZV 200 μl 50 μl total NA external lysis

Respiratory material; swabs and lavages

resuspend dry swabs in 1 ml transport medium

EV, HPeV, HRV, InfA, InfB, HAV, RSV, hMPV, PIV 1-4, hCoV, HBoV

200 μl 50 μl total NA external lysis

Faeces pre-lysis of 50 μl faeces in 500 μl MP lysisbuf-fer; Spin down and use supernatant as input

EV, HPeV 50 μl 50 μl total NA external lysis

Biopsy O/N pre-lysis in 200 μl 1% SDS/prot K solution x 56°C; spin downand use 100 μl as input

HAV, CMV 100 μl 50 μl total NA external lysis

Culture supernatant none any culturable virus 20 μl 50 μl total NA external lysis

Vesicle fluid none HSV, VZV 20 μl 50 μl total NA external lysis

Urine none BK, CMV 200 μl 50 μl total NA external lysis

Materials and Methods

Virus stocks and patient material DNA viruses Human Adenovirus (HAV) and Cytomegalovirus(CMV), as well as RNA viruses Human Parechovirus(HPeV) and Influenza A virus, were isolated and cultured from sample materials sent to the laboratory for molecular diagnosis. Virus stocks were TCID50* quantified.

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*Median tissue culture infective dose; the amount of a cytopathogenicagent (virus) that will produce a cytopathic effect in 50% of cell culturesinoculated, expressed as TCID50/ml.

Testing requires efficient and convenient nucleic acid extraction protocols. Due to the increased size of sample flow, nucleic acid extraction is preferably automated to pro-cess many samples, in as little time as possible. For this, there is a need for a fast, automated high-throughput purifi-cation system that can handle a variety of materials, prefera-bly in the same extraction run and using the same extraction protocol. To investigate if the MagNA Pure 96 System is able to efficiently extract nucleic acid from various sample mate-rials, we spiked different clinical materials with various amounts of different characterized virus stocks. Internal controls were added to all samples to monitor extraction efficiency of the MagNA Pure 96 System. The readout was performed using real-time PCR with hydrolysis probes in a Roche LightCycler ®480 Real Time PCR Instrument.

Table 1: Treatment of various human sample materials to be extracted in the MagNA Pure 96 System in the AMC diagnostic laboratory.

Cytomegalo Virus (CMV), Herpes Simplex Virus (HSV), Varicella Zoster Virus (VZV), Epstein Bar Virus (EBV), Human Herpes Virus 8 (HHV8), Entero Virus (EV), Human parEcho Virus (HPeV), Human Rhino Virus (HRV), Influenza A/B (InfA/B), Human Adeno Virus (HAV), Respiratory Syncytial Virus (RSV), Human Metapneumo Virus (hMPV), Para Influenza Virus (PIV), Human Corona Virus (hCoV), Human Boca Virus (HBoV)

** Final input volume of lysed sample in the MagNA Pure 96 System is 500 μl for all materials

Materials and Methods

Cerebrospinal fluid (CSF), EDTA whole blood, plasma,faeces, throat swabs and adenoid tissue biopsies were takenfrom the Academic Medical Center clinical virology sample archives. All human sample materials used were collected according to general hospital standards. We have tested the daily routine using the MagNA Pure 96 System in our lab by spiking the samples with viral targets prior to extraction. Sample types analyzed in our laboratory are summarized in Table 1. We used different input volumes of samples, according to the sample type in order to make simultaneous extraction of a variety of sample types possible.

PCR Controls Positive controls were purified plasmids containing the amplicon sequence of interest. In addition, two differentIn-ternal Controls (IC) were used. Phocine Herpes virus (PhoHV plasmid) for DNA targets1 and Equine Arteritis virus (EAV) for RNA targets. 2 Sample Pretreatment • Cerebrospinal Fluid (CSF), EDTA anticoagulated whole

blood and plasma were used without additionalpretreatment.

• Throat swabs were resuspended in 2 ml of virustransportation medium (UTM Universal TransportMedium, Copan).

• Faeces was pre-treated as follows: 50 μl faeces wasadded to 450 μl MagNA Pure 96 lysis buffer, mixed, and subsequently incubated for 10 minutes at roomtemperature (+15 to +25°C). The suspension wascentrifuged at 13,000 x g and the supernatant was spiked with target virus and IC prior to extraction.

• Tissue biopsies (ca. 20-40 mm3 in size) were pretreated asfollows: Freshly isolated adenoid tissue was submerged in1 ml of RNAlater (Ambion) and stored at -15 to -25°C.Prior to use for extraction RNAlater was pipetted fromthe (solid) tissue which was subsequently washed withPBS; 200 μl of a 1 mg/ml Proteinase K/1% SDS solutionwas added to the tissue, and the mixture was incubatedovernight at 56°C. The tube was centrifuged at 13,000 x gand 100 μl of the supernatant was spiked with InternalControl (IC), added to 400 μl MagNA Pure 96 lysis bufferand used for extraction.

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DNA/RNA isolation using MagNA Pure 96 System One single extraction protocol was used for all the different sample types. All extractions were performed using the MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche Diagnostics), in combination with the Viral NA Plasma SV external lysis protocol. For all materials, the input volume of lysed sample was set to 500 μl and elution was set to 50 μl. Unless otherwise stated, the guidelines from the package insert were followed.To assess the MagNA Pure 96 System utility, multiplex PCR using the LightCycler® 480 Instrument was used to analyze MagNA Pure 96-purified DNA/RNA or, alternatively, in case of viral RNA targets, subjected to reverse transcription (RT)-reaction prior to PCR, as described previously.3,4 In brief, 40 μl of the MagNA Pure 96 System eluate (80% of the original input) was mixed with 10 μl RT-reaction mix, consisting of 1,500 ng of hexamers, 1x CMB1 buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 0.1% Triton X-100), 0.4 U of reverse transcriptase per μl, 120 μM (each) deoxynucleoside triphosphate, 0.08 U of RNAsin per μl, and 5 mM MgCl2. The mixture was incubated for 30 min at 42°C.

Real-Time PCR Amplification and Detection(using TaqMan® Probes) As input in PCR, either 5 μl of the MagNA Pure 96System eluate (10% of the original input) for DNA or5 μl of the RT-reaction (8% of the original input) forRNA was used in a final volume of 20 μl. In all PCRs performed, the Roche LightCycler® 480 Probes Master (containing a hot start Taq enzyme) was used supplemented with 1U/ml Uracil-DNA Glycosylase (UNG), 900 nM of each primer and 200 nM of each TaqMan® probe. Sequences of the specific primers and probes used are published elsewhere. 5,6,7

Detection was done using the Roche LightCycler® 480Real-Time PCR Instrument. For all targets the samePCR program was used with the following cyclingconditions: 2 min x 50°C (activation UNG), 10 min x 95°C(denaturation template and activation polymerase),followed by 45 rounds consisting of 15 sec x 95°Cand 1 min x 60°C.

For data analysis with the LightCycler ® 480 Software,the Second Derivative Maximum analysis method was used.

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Results

Plasma

Cycles

Fluo

resc

ence

(48

3–53

3)

21,890 -

19,890 -

17,890 -

15,890 -

13,890 -

11,890 -

9,890 -

7,890 -

5,890 -

3,890 -

1,890 -

-0,110 -

- - - - - - - - - - - - - - - - - - - - - -

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

EDTA whole blood

Cycles

Fluo

resc

ence

(48

3–53

3)

19,202 -

17,702 -

16,202 -

14,702 -

13,202 -

11,702 -

10,202 -

8,702 -

7,202 -

5,702 -

4,202 -

2,702 -

1,202 -

-0,298-

- - - - - - - - - - - - - - - - - - - - - -

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

CSF

Cycles

Fluo

resc

ence

(48

3–53

3)

32,677 -

29,677 -

26,677 -

23,677 -

20,677 -

17,677 -

14,677 -

11,677 -

8,677 -

5,677 -

2,677 -

-0,323 -

- - - - - - - - - - - - - - - - - - - - - -

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

Throat swabs

Cycles

Fluo

resc

ence

(48

3–53

3)

2,635 -

2,435 -

2,235 -

2,035 -

1,835 -

1,635 -

1,435 -

1,235 -

1,035 -

0,835 -

0,635 -

0,435 -

0,235 -

0,035 -

- - - - - - - - -

5 10 15 20 25 30 35 40 45

Figure 1a. Shows the amplification curves for CMV (FAM), PhoHV IC (HEX) in inset. Plasma. 200 μl plasma was spiked with CMV (dilution series 1 x 104 to 1 x 102 copies and negative control) and PhoHV IC (2.5 x 103 copies). After extraction the DNA was directly analyzed in real-time PCR.

Figure 1b. Shows the amplification curves for CMV(FAM), PhoHV IC(HEX) in inset. EDTA whole blood. 50 μl EDTA whole blood was spiked with CMV dilutionseries (1x 104 to 1 x 102 copies and negative control) and PhoHV IC (2.5 x 103 copies). After extraction the DNA was directly analyzed in real-time PCR.

Figure 1c. Shows the amplification curves for HPeV (FAM), EAV IC(HEX) in inset. CSF. 200 μl CSF was spiked with HPeV dilution series (1 x 105 to 1 x 101 copies and negative control) and EAV-IC (2 x 103 copies). After extraction the RNA was subjected to RT-reaction and real-time PCR.

Figure 1d. Shows the amplification curves for HAV (LC670), HPeV (FAM;upper inset) and PhoHV IC (HEX; lower inset) are also shown. Throat swabs. A 200 μl throat swab sample was spiked with Human Adeno Virus dilution series (1 x 108, 1 x 107, 1 x 105, 1 x 103 and 1 x 101 copies and negative control), HPeV fixed concentration(1 x 103 copies) and PhoHV IC (2.5 x 10 copies). After extraction the nucleic acid was subjected to RT-reactionand real-time PCR.

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Discussion

We show in this paper that the MagNA Pure 96 System isable to extract nucleic acid, RNA and/or DNA, fromdifferent kinds of clinical samples. Furthermore, a varietyof sample types could be extracted efficiently in the samerun using the same extraction protocol. To perform theseexperiments, we selected samples that had been tested as negative in our diagnostic department. These samples were spiked with various loads of different viruses, and subsequently extracted (external lysis protocol) and used for detection. To all samples, an internal control was added to exclude inhibition effects and monitor extraction and PCR efficiencies.

Discussion Detection using the Roche LightCycler® 480 Real-Time PCR Instrument revealed that extraction from all six different types of sample materials tested was very efficient. Even with known difficult materials, such as faeces and biopsies, the MagNA Pure 96 System performed well.

In addition, we extracted nucleic acid from 2 differentvolume inputs (50 and 200 μl) of EDTA whole blood frompatients positive for CMV. Higher inputs led to highersensitivity in 7/8 samples, crossing point (Cp) cycle valueswere approximately 1.7 cycle lower while Cp values forIC remained similar (data not shown).

Results

Faeces

Cycles

Fluo

resc

ence

(48

3–53

3)

30,232 -

27,732 -

25,232 -

22,732 -

20,232 -

17,732 -

15,232 -

12,732 -

10,232 -

7,732 -

5,232 -

2,732 -

0,232 -

- - - - - - - - - - - - - - - - - - - - - -

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44

EDTA whole blood

Cycles

Fluo

resc

ence

(48

3–53

3)

17,023 -

15,523 -

14,023 -

12,523 -

11,023 -

9,523 -

8,023 -

6,523 -

5,023 -

3,523 -

2,023 -

0,523 -

- - - - - - - - -

5 10 15 20 25 30 35 40 45

Figure 1e. Shows the amplification curves for HPeV (FAM), EAV IC (HEX) in inset. Faeces. Faeces (50 μl in bouillon) was pre-lysed in 500 μl MagNA Pure lysis buffer for 10 min at room temperature (+15 to +25°C). Samples were spun down for 2 minutes. Supernatants were spiked with a HPeV dilution series(1 x 105 to 1 x 101 copies and negative control) and EAV IC (2 x 103 copies). After extraction, the RNA was subjected to RT-reaction and real-time PCR.

Figure 1f. Shows the amplification curves for HAV (LC670), PhoHV IC(HEX) in inset. Biopsies. 6 adenoid biopsies, 4 positive and 2 negative for HAV, storedat -15 to -25°C in RNAlater, underwent pretreatment, as described above; 100 μl of the biopsy material was spiked with PhoHV IC (2.5 x 103 copies). For extraction, 100 μl of the samples was used in the MagNA Pure 96 System.After extraction, the DNA was directly analyzed using real-time PCR.

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Conclusion

The MagNA Pure 96 System, in combination with theMagNA Pure 96 DNA and Viral NA Small Volume Kit, is ahighly efficient and fast system for the extraction of nucleicacid from a variety of clinical sample materials. Remarkably,a single extraction protocol combined with a singledownstream real-time RT-PCR protocol can be used. TheMagNA Pure 96 System allows for high-throughput and fast

turnaround times (it takes less than 60 minutes to extractnucleic acid from 96 samples). Due to the use of a singlegeneric extraction protocol different materials can beextracted together simultaneously, thereby adding to theflexibility of the system. This is especially useful formolecular diagnostic laboratories that handle large andheterogeneous sample workflows.

References

1. Van Doornum et al. Journal of Clinical Microbiology, Februari 2003, Vol. 41 no.2; 576

2. Scheltinga et al., Journal of Clinical Virology, August 2005, Vol. 33 no. 4; 306

3. Beld et al. Journal of Clinical Microbiology, July 2004, Vol. 42 no. 7; 3059

4. Molenkamp et al. Journal of Virological Methods, May 2007, Vol. 141 no. 2; 205

5. Benschop et al, Journal of Clinical Virology, Februari 2008, Vol. 41 no.2; 69

www.ehec.org/pdf/Laborinfo_01062011.pdf

6. Damen et al. Journal of Clinical Microbiology. December 2008. Vol. 46 no.12; 3997.

7. Jansen et al. Journal of Clinical Virology, May 2011, Epub ahead of print

Important Note: Regarding the specific assay(s) described, Roche was neither involved in establishing the experimental conditions nor in defining the criteria for the performance of the specific assays. Roche therefore cannot take any responsibility for performance or interpretation of results obtained for the bio-logical target parameter(s) described by the authors or otherusers using a similar experimental approach.

Potential users are informed to be aware of and in accordance with local regulations for assay validation and the scope of use for the involved Roche products, and to ensure that their use is valid in the countries where the experiments are per-formed.

The MagNA Pure 96 Instrument (06 541 089 001) is for in vitro diagnostic use.

The LightCycler® 480 Instrument is for life science research only. Not for use in diagnostic procedures.

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