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Comparison of manual and automated nucleic acid isolation methods for

HBV-DNA and HCV-RNA assays

Article  in  Le infezioni in medicina: rivista periodica di eziologia, epidemiologia, diagnostica, clinica e terapia delle patologie infettive · September 2015

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Le Infezioni in Medicina, n. 3, 247-252, 2015

Articolo originale / Original article 247

Comparison of manual and automated

nucleic acid isolation methods
for HBV-DNA and HCV-RNA assays
Confronto di metodi manuali e automatizzati di isolamento per i test
HBV-DNA e HCV-RNA

Gulhan Yagmur1, Hatice Uludag Altun2, Selma Gökahmetoglu3, Ela Basok3

1
Council of Forensic Medicine, Postmortem Microbiology, Istanbul, Turkey;
2
Turgut Ozal University Faculty of Medicine, Department of Medical Microbiology, Ankara, Turkey;
3
Erciyes University Medical Faculty, Department of Microbiology, Kayseri, Turkey

n INTRODUCTION performed using automated isolation devices, as

well as manual procedures. NucliSens easyMAG

H BV and HCV infections are important public

health issues both in Turkey and through-
out the world due to their complications in the
isolation device (bioMérieux, France) is an auto-
mated system ensuring nucleic acid isolation from
the clinical samples, based on the silica extraction
advanced period [1]. The techniques for nucleic technology [2, 7]. The present study aimed to com-
acid extraction that may affect the sensitivity of pare the automated isolation system “NucliSens
the polymerase chain reaction (PCR) method easyMAG” (bioMérieux, France) with the conven-
used to diagnose and to monitor these infections tional manual isolation method (Qiagen, Germa-
are increasingly employed in clinical laboratories ny) for HBV-DNA and HCV-RNA detection.
due to their high sensitivity and specificity [2, 3].
HBV-DNA quantitation is important to determine
the level of viral replication, to stage the infection, n MATERIAL AND METHODS
to monitor the response to the treatment, and to
establish the occurrence of antiviral resistance; Samples
HCV-RNA quantitation is also important to moni- The evaluation was carried out from patient
tor the response to the treatment [1]. Therefore, samples submitted to the Department of Virology
the fast and proper performance of the viral nu- Laboratory, Erciyes University, Kayseri, Turkey.
cleic acid isolation used in clinical laboratories is These serum samples were stored in the Eppen-
critical for diagnosing and monitoring the patient. dorf tubes at -80°C until the time of study. The
Viral nucleic acid isolation has been performed study included 93 serum samples, 49 were for
manually in many laboratories until recently; HBV-DNA assay, and 44 were for HCV-RNA as-
however, there have been important advances in say. DNA and RNA extraction from the samples
the automated isolation systems in recent years was performed using the automated isolation
[4]. Conventional manual extractions are incon- system NucliSens easyMAG (bioMérieux, France)
venient methods that are open to contamination and the manual method QIAmpMin Elute Kit
[5, 6]. Therefore, nucleic acid isolation is currently (Qiagen, Germany). The amplification process of
the nucleic acids obtained through both methods
was conducted in the iCycler device (Bio-Rad,
Corresponding author USA) with the PCR method using the “Fluorion
Gulhan Yagmur HBV QNP” and “Fluorion HCV QNP” kits (Ion-
[email protected] tek, Turkey).
248 G. Yagmur, et al.

Manual extraction Statistical analysis

DNA and RNA isolation from the serum samples The compliance between two methods was evalu-
was performed using the “QIAmpMin Elute Kit” ated using Kendall’s tau-c and Kappa statistical
(Qiagen, Germany) following the manufacturer’s analysis in both tests.
recommendations. Twenty-five µL proteinase K
and 200 µL plasma were placed into each tube.
A mixture of 220 µL buffer Al and 6.2 µL carrier n RESULTS
RNA was prepared per sample, and 200 µL of this
mixture was added to the tube, and then the tube No amplification was found in the two methods in
was incubated at 56°C for 15 minutes. The col- 10 (20.4%) of 49 samples for HBV-DNA. Two (4%)
lection tubes were centrifuged at 8000 rpm for 1 samples were found to be <20 IU/ml (negative)
minute. Five hundred µL buffer was added to the by the manual method, whereas this value was
tube, and then the procedure was repeated. The <100 IU/ml by the easyMAG isolation method;
colons were placed into the clean collection tubes six samples (12.2%) were found to be <100 IU/ml
and then centrifuged again at 12,500 rpm for 3 and <20 IU/ml (negative) by the manual method
minutes. Forty µL buffer was added to the tubes, and easyMAG isolation method, respectively. The
and then the tubes were kept at room temperature values of 11 (22.4%) samples, for which quantita-
for one minute and centrifuged at 14,000 rpm for tive results were obtained by both methods were
one minute. The nucleic acid samples collected in observed to be consistent with one another. There
the lower tube were used for PCR. was a significant logarithmic difference in HBV-
DNA quantitation for 20 samples. It was found
Extraction with NucliSens easyMAG that the numbers of nucleic acid were higher in 17
DNA and RNA isolation from the samples was (34.6%) of 20 samples by the easyMAG isolation
performed using the NucliSens easyMAG au- method compared to the manual method, and the
tomated isolation system (bioMérieux, France) amount of nucleic acid was higher in three (6.1%)
following the manufacturer’s recommendations. samples by the manual method compared to the
Two hundred and fifty µL was taken from the easyMAG isolation method. In conclusion, for
samples that would be used in extraction, and the HBV-DNA assay, compliance was found in 21
was distributed into each well. Two µL lysis buf- (42.8%) samples; nine (18.3%) samples had a high-
fer was added to them and mixed, then incubated er amount of viral nucleic acid with the manual
at room temperature for 10 minutes. In an empty method, whereas 19 samples (38.7%) were found
tube, 550 µL internal control and 550 µL magnetic to have a higher amount of nucleic acid with the
silica were mixed. Then, 100 µL was taken from automated system (Table 1).
this mixture and distributed onto the samples. No amplification was found by the two methods
The device was operated and the extraction pro- in 12 (27.2%) of 44 samples for HCV-RNA. Nine
cess was initiated. The obtained nucleic acids (20.4%) samples were found to be <700 IU/mL
were used for PCR. (negative) by the easyMAG isolation method,
whereas this value was <5000 IU/ mL by the
Amplification process manual method; four (9%) samples were found to
The amplification process of the DNA and RNA be <5000 IU/ mL (positive) by both methods. The
obtained through both methods was conducted values of 12 (27.2%) samples for which quantita-
and determined in the iCycler device (Bio-Rad, tive results were obtained by both methods and of
USA) with the PCR method using the “Fluorion three (6.8%) samples >106 IU/mL were observed
HBV QNP” and “Fluorion HCV QNP” kits (Ion- to be consistent with one another. A logarithmic
tek, Turkey). For the test used in quantitatively difference of significance was found in the HCV-
detecting HBV-DNA, the dynamic range was RNA quantitation for four samples. It was also
1x102-106 IU/mL, and the analytical sensitivity found that the amount of nucleic acid obtained
limit was 20 IU/mL. For the test used to quantita- by the manual method was higher in three (6.8%)
tively detection of HCV-RNA, the dynamic range of four samples compared to the easyMAG isola-
was 5x103-1x106 IU/mL, and the analytical sensi- tion method, and one (2.2%) sample had a higher
tivity limit was 700 IU/mL. number of nucleic acid by the easyMAG isolation
 Comparison of manual and automated nucleic acid isolation methods for HBV-DNA and HCV-RNA assays 249

Table 1 - EasyMAG isolation system vs. manual isolation method for HBV-DNA quantitation (n=49).
Isolation Manual isolation
Total
with Easymag N/A <100 IU/mL 102 IU/mL 103 IU/mL 104 IU/mL 105 IU/mL 106 IU/mL 107 IU/mL
N/A 10 6 0 0 0 0 0 0 16
<100 IU/ mL 2 5 0 1 0 0 0 0 8
102 IU/mL 1 2 0 0 0 0 0 0 3
103 IU/mL 0 1 2 2 0 2 0 0 7
104 IU/mL 0 0 0 2 0 0 0 0 2
105 IU/mL 0 0 0 0 3 0 0 0 3
106 IU/mL 0 0 0 0 0 3 1 0 4
107 IU/mL 0 0 0 0 0 0 3 3 6
Total 13 14 2 5 3 5 4 3 49
N/A: Not Applicable.

method compared to the manual method. In con- DNA has gained importance upon the knowledge
clusion, for the quantitative HCV-DRA assay, to- that people with negative serological markers
tal compliance was found in 31 (70.4%) samples; may also carry and transmit HBV. Furthermore,
12 (27.2%) samples had a higher amount of viral investigating HBV-DNA is important to dem-
nucleic acid with the manual method, whereas onstrate the presence of replication in HBsAg-
one sample (2.2%) was found to have a higher positive cases [8]. For HCV-RNA assays through
number of nucleic acid with the automated sys- molecular tests, the quantitative tests in particular
tem (Table 2). provide the amount of virus in the patient’s blood
Nucleic acid isolation was achieved in a short in international units and are used to monitor the
time, as 40 minutes from 24 patient samples with disease prognosis [9].
the easyMAG isolation device, whereas this time Nucleic acid isolation, which is the first step of the
was up to 90 minutes with the manual isolation molecular methods, is performed manually, takes
method. a long time, and requires caution due to the risk of
Quantitative results from the isolation of HBV- contamination. After the semi- or fully-automat-
DNA and HCV-RNA by manual and automated ed nucleic acid isolation systems have come into
systems were found to be statistically consistent routine use in laboratories in the recent years, the
(kappa value 0.40-0.60, moderate agreement). effort is to ensure reduced rates of contamination
and avoidance of technical errors [10-12].
The real-time PCR test, among the commonly
n DISCUSSION used molecular tests, has a high sensitivity, and
therefore if a small contamination occurred dur-
The most common conventional method used for ing the isolation, this may lead to false positiv-
diagnosing HBV and HCV infections is the ELI- ity. Loens et al. reported in their study compar-
SA test. Currently, molecular tests as well as the ing NucliSens easyMAG and the manual method
ELISA test are frequently used. Analysis of HBV- that they did not obtain any false positive results

Table 2 - EasyMAG isolation system vs. manual isolation method for HCV-RNA quantitation (n=44).
Isolation Manual isolation
Total
with Easymag N/A <5000 IU/mL 103 IU/mL 104 IU/mL 105 IU/mL 106 IU/mL
N/A 12 9 0 0 0 0 21
<5000 IU/mL 0 4 0 0 0 0 4
103 IU/mL 0 1 2 0 0 1 4
104 IU/mL 0 0 0 6 0 0 6
105 IU/mL 0 0 0 0 4 2 6
106 IU/mL 0 0 0 0 0 3 3
Total 12 14 2 6 4 6 44
N/A: Not Applicable.
250 G. Yagmur, et al.

[2]. Furthermore, the present study found that the MAG automated system was found to detect vi-
number of very low viral loads obtained from the ral loads at a higher rate compared to the manual
manual method in total 15 samples were nega- method, especially in samples with a high viral
tive by the NucliSens easyMAG isolation method, load. It is considered that this cannot be ignored
supporting this result. in the HBV-DNA and HCV-RNA assays, where
The studies of the NucliSens easyMAG automat- the amount of viral load is important, especially
ed system versus the manual method reported in the diagnosis, treatment, and follow-up. Fur-
that the automated system was more sensitive in thermore, the cut-off value for HCV-RNA may be
nucleic acid multiplication, detected the amount not low enough for accurate detection. Similarly,
of DNA at a higher level than the manual method, lower viral loads may cause inaccurate detection
was easy to use, and produced results in a short that may affect the comparison. In addition, our
time (40 minutes) [13-16]. However, Soetens et al. low number of serum samples may not be consid-
concluded in their CMV-DNA isolation study that ered to provide very reliable results for compari-
the manual method produced better results com- son of the extraction methods.
pared to the automated method; however, the au- In conclusion, automated nucleic acid isolation
tomated system was more advantageous due to methods have greater costs than the manual meth-
its automation feature[17]. In the present study, ods; however, they can be used with confidence in
the manual method was found to detect viral routine laboratories due to their advantages, such
loads at a higher rate compared to the NucliSens as providing standardization during the proce-
easyMAG automated system, in only six samples. dure, a low rate of contamination, being fast, and
In the studies of the NucliSens easyMAG auto- running multiple samples at the same time.
mated system versus other automated systems,
a high rate of consistency was found among the Conflict of interests
systems and it was concluded that these systems We declare that we have no conflict of interest.
were more advantageous for detecting viral loads
compared to the manual method [18-22]. Addi- Keywords: Nucleic acid isolation, HBV-DNA,
tionally, in the present study, the NucliSens easy- HCV-RNA, NucliSens easyMAG.

SummaRY
In the diagnosis and monitoring of hepatitis B virus compliance was found in 21 (42.8%) samples in the
(HBV) and hepatitis C virus (HCV) infections, it is im- HBV-DNA assay; nine (18.3%) samples had a higher
portant to use methods that can provide rapid and reli- amount of viral nucleic acid with the manual meth-
able results. The present study aimed to compare the od, whereas 19 samples (38.7%) were found to have
automated and manual extraction methods during the a higher amount of nucleic acid with the automated
nucleic acid isolation phase for HBV-DNA and HCV- system. For the HCV-RNA assay, total compliance was
RNA assays. found in 31 (70.4%) samples; 12 (27.2%) samples had
The study included 93 serum samples, 49 of which a higher amount of viral nucleic acid with the manual
were for the HBV-DNA assay and 44 for the HCV- method whereas one sample (2.2%) was found to have
RNA assay. DNA and RNA isolation from the samples a higher amount of nucleic acid with the automated
was performed manually with a “QIAmpMin Elute system. It was concluded that the NucliSens easy-
Kit” (Qiagen, Germany) and the automated isolation MAG automated isolation system can be used with
system, NucliSens easyMAG (BioMérieux, France). confidence for nucleic acid extraction due to its higher
All the extraction products were amplified using the sensitivity, providing results in a shorter time, and as-
iCycler device (Bio-Rad, USA). With both methods, sured standardization.
 Comparison of manual and automated nucleic acid isolation methods for HBV-DNA and HCV-RNA assays 251

RIASSUNTO
Nella diagnosi e nella gestione delle infezioni da virus dell’e- no un maggiore quantitativo di acido nucleico virale con il
patite B (HBV) e da virus dell’epatite C (HCV), è importan- metodo manuale, mentre per 19 campioni (38,7%) è stato
te avvalersi di metodiche in grado di offrire risultati rapidi rilevato un quantitativo di acido nucleico maggiore con il
e affidabili. Il presente studio è volto a confrontare i metodi sistema automatizzato.
automatizzati e manuali nella fase di estrazione dell’acido Per il test HCV-RNA, i risultati sono stati sovrapponibili
nucleico nei test HBV-DNA e HCV-RNA. Nello studio sono in 31 (70,4%) campioni; 12 (27.2%) campioni presentava-
stati presi in considerazione 93 campioni di siero, di cui 49 no un maggiore quantitativo di acido nucleico virale con il
da sottoporre a test HBV-DNA e 44 a test HCV-RNA. metodo manuale, mentre per un campione (2,2%) è stato
L’isolamento di DNA e RNA dai campioni è stato effettuato rilevato un quantitativo di acido nucleico maggiore con il
manualmente con “QIAmpMin Elute Kit” (Qiagen, Ger- sistema automatizzato.
mania) e con il sistema di isolamento automatizzato Nucli- I risultati osservati consentono di concludere che il sistema
Sens easyMAG (BioMérieux, Francia). Tutti i prodotti di di isolamento automatizzato NucliSens easyMAG può esse-
estrazione sono stati amplificati utilizzando il dispositivo re considerato un metodo affidabile per l’estrazione di acido
iCycler (Bio-Rad, Stati Uniti). Con entrambi i metodi, i nucleico in virtù della sua più elevata sensibilità, in grado di
risultati sono stati sovrapponibili in 21 (42,8%) campioni fornire risultati in tempi più brevi e garantire la standardiz-
testati per HBV-DNA; nove (18,3%) campioni presentava- zazione della procedura.

n REFERENCES lecular, clinical and diagnostic virology (Ustacelebi S., Aba-

cioglu H., Badur S., Eds) 2004, 203-209. Günes Kitabevi
[1] Sayiner A. Viral load determination, In: 2nd Molecu- Press, Ankara.
lar, clinical and diagnostic virology course book (Ustacelebi [10] Alp A., Us D., Hascelik G. Comparison of manual
S., Ed) 2006, 296-297. Ankara Microbiology Society, An- and automated (Magna Pure) nucleic acid isolation
kara. methods in molecular diagnosis of HIV infections. Mik-
[2] Loens K., Bergs K., Ursi D., Goossens H., Ieven M. robiyol. Bul. 38, 1-2, 77-83, 2004.
Evaluation of nuclisens easyMAG for automated nu- [11] Alp A., Alp S., Sarıbas Z., Hascelik G. Comparison
cleic asit extraction from various clinical specimens. J. of three different mycobacterial DNA isolation meth-
Clin. Microbiol. 45, 2, 421-425, 2007. ods. Mikrobiyol. Bul. 41, 3, 395-402, 2007.
[3] Tang Y.W., Sefers S.E., Li H., Kohn D.J., Procop G.W. [12] Alp A., Hascelik G. Comparison of 3 nucleic acid
Comparative evaluation of three commercial systems isolation methods for the quantification of HIV-1 RNA
for nucleic acid extraction from urine specimens. J. Clin. by Cobas Taqman real-time polymerase chain reaction
Microbiol. 43, 9, 4830-4833, 2005. system. Diagn. Microbiol. Infect. Dis. 63, 4, 365-371, 2009.
[4] Fiebelkorn K.R., Lee B.G., Hill C.E., Caliendo A.M., [13] Dundas N., Leos N.K., Mitui M., Revell P., Rogers
Nolte F.S. Clinical evaluation of an automated nucleic B.B. Comparison of automated nucleic acid extraction
acid isolation system. Clin. Chem. 48, 9, 1613-1615, 2002. methods with manual extraction. J. Mol. Diagn. 10, 4,
[5] Beuselinck K., Ranst M.V., Eldere J.V. Automated 311-316, 2008.
extraction of viral pathogen RNA and DNA for high- [14] Faucher B., Miermont F., Ranque S., Franck J., Piar-
throughput quantitative Real-time PCR. J. Clin. Micro- roux R. Optimization of Toxoplasma gondii DNA extrac-
biol. 43, 11, 5541-5546, 2005. tion from amniotic fluid using NucliSENS easyMAG
[6] Knepp J.H., Geahr M.A., Forman M.S., Valsamiks and comparison with QIAamp DNA minikit. Eur. J.
A. Comparison of automated and manual nucleic acid Clin. Microbiol. Infect. Dis. 31, 6, 1035-1039, 2012.
extraction methods for detection of enterovirus RNA. J. [15] Perandin F., Pollara P.C., Gargiulo F., Bonfanti C.,
Clin. Microbiol. 41, 8, 3532-3536, 2003. Manca N. Performance evaluation of the automated
[7] Jeddi F., Piarroux R., Mary C. Application of the Nu- NucliSens easyMAG nucleic acid extraction platform in
cliSENS easyMAG system for nucleic acid extraction: comparison with QIAamp Mini kit from clinical speci-
optimization of DNA extraction for molecular diagno- mens. Diagn. Microbiol. Infect. Dis. 64, 2, 158-165, 2009.
sis of parasitic and fungal diseases. Parasite 20, 52, 2013. [16] Ginocchio C.C., Manji R., Lotlikar M., Zhang F.
[8] Badur S. Viral Hepatitis (HAV, HBV, HDV), In Mo- Clinical evaluation of NucliSENS magnetic extraction
lecular, clinical and diagnostic virology (Ustacelebi S., and NucliSENS analyte-specific reagents for real-time
Abacioglu H., Badur S., Eds) 2004, pp 175-202. Günes detection of human metapneumovirus in pediatric re-
Kitabevi Press, Ankara. spiratory specimens. J. Clin. Microbiol. 46, 4, 1274-1280,
[9] Ustacelebi S., Ergünay K., Hepatitis C Virus, In Mo- 2008.
252 G. Yagmur, et al.

[17] Soetens O., Vauloup-Fellous C., Foulon I., et al. [20] Pillet S., Bourlet T., Pozzetto B. Comparative eval-
Evaluation of different cytomegalovirus (CMV) DNA uation of the QIAsymphony RGQ system with the
PCR protocols for analysis of dried blood spots from easyMAG/R-gene combination for the quantitation of
consecutive cases of neonates with congenital CMV in- cytomegalovirus DNA load in whole blood. Virol. J. 9,
fections. J. Clin. Microbiol. 46, 3, 943-946, 2008. 9, 231, 2012.
[18] Chan K.H., Yam W.C., Pang C.M., et al. Comparison [21] Shulman L.M., Hindiyeh M., Muhsen K., Cohen
of the NucliSens easyMAG and Qiagen BioRobot 9604 D., Mendelson E., Sofer D. Evaluation of four different
nucleic acid extraction systems for detection of RNA systems for extraction of RNA from stool suspensions
and DNA respiratory viruses in nasopharyngeal aspi- using MS-2 coliphage as an exogenous control for RT-
rate samples. J. Clin. Microbiol. 46, 7, 2195-2199, 2008. PCR inhibition. PLoS One 7, e39455, 2012.
[19] Lee A.V., Atkinson C., Manuel R.J., Clark D.A. [22] Laakso S., Kirveskari J., Tissari P., Mäki M. Evalu-
Comparative evaluation of the QIAGEN QIAsympho- ation of high-throughput PCR and microarray-based
ny® SP system and bioMérieux NucliSens easyMAG assay in conjunction with automated DNA extraction
automated extraction platforms in a clinical virology instruments for diagnosis of sepsis. PLoS One 6, e26655,
laboratory. J. Clin. Virol. 52, 4, 339-343, 2011. 2011.

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