Complete article - Revue de Médecine Vétérinaire

172
DIMITROV (K.) AND COLLABORATORS
RNA extraction for molecular detection of Newcastle
disease virus – comparative study of three methods
K. DIMITROV1*, A. CLAVIJO2, L. SNEED2
National Diagnostic and Research Veterinary Medical Institute, Bulgaria, Aksakovo, 9154, 2 Batova Street.
Texas Veterinary Medical Diagnostic Laboratory, USA, College Station, TX 77841-3040, PO Box Drawer 3040
1
2
*Corresponding author: [email protected]
SUMMARY
RESUME
RNA extraction is one of the essential factors influencing the sensitivity of
real-time reverse transcription polymerase chain reaction (rRT-PCR) as a
diagnostic test for Newcastle disease virus (NDV). The separation of RNA
from the other biological substances present in the clinical sample and
subsequent removal of reagent residues are particularly important because
of the possible inhibitory effect on the PCR. The aim of the present study
was to compare the sensitivity of rRT-PCR following Newcastle disease virus
RNA extraction with Qiagen® RNeasy Mini Kit, Mag MAX™-96 AI/ND Viral
RNA Isolation Kit and TRIZOL® LS. The length of time required to carry
out the extraction of a certain number of samples as well as the presence of
any cross contamination was also evaluated in this study. A set of ten avian
paramyxovirus 1 (APMV 1) samples provided by the National Veterinary
Services Laboratories – United States Department of Agriculture, Ames,
Iowa was used for this comparison. For each sample, RNA was extracted in
triplicate using each of the three methods. The isolated RNAs were tested
by Matrix-gene rRT-PCR using the AgPath-ID™ One-Step RT-PCR and the
mean Ct values were analyzed statistically. The lowest Ct values in the rRTPCR were observed using the Qiagen® RNeasy Mini Kit. Mag MAX™-96
AI/ND Viral RNA Isolation Kit in combination with an automated system
allowed processing of a greater number of samples in a shorter time period,
which would be especially beneficial during a Newcastle disease outbreak.
Cross contamination was not observed for any of the utilized methods.
Comparaison de trois méthodes d’extractions des ARN pour le diagnostic
de la maladie de Newcastle
Keywords : RNA, extraction, APMV 1, NDV, Newcastle
disease, rRT-PCR
L’extraction des ARN est l’un des facteurs essentiels qui influencent la
sensibilité de détection du virus de la maladie de Newcastle par la technique
de RT- PCR. La séparation de l’ARN à partir d’autres substances biologiques
présentes dans l’échantillon clinique et l’élimination ultérieure des résidus
de réactifs sont particulièrement importants en raison d’effets inhibiteurs
possible sur la réaction. Le but de cette étude était de comparer la sensibilité
de lors de l’utilisation de trois kits d’extraction : Qiagen® RNeasy Mini Kit,
Mag MAX™-96 AI/ND Viral RNA Isolation Kit and TRIZOL® LS. La durée de
temps nécessaire pour l’extraction des échantillons ainsi que la présence de
contamination croisée a également été évaluée. Un ensemble dix échantillons
de paramyxovirus aviaire 1 ( PMV 1 ) fournis par le Laboratoire national
des Services vétérinaires – (United States Department of Agriculture, Ames,
Iowa) a été utilisé pour cette comparaison. Pour chaque échantillon, l’ARN
a été extrait en triple exemplaire à l’aide de chacune des trois méthodes.
Les ARN isolés ont été testés par Matrix-gene rRT-PCR en utilisant the
AgPath-ID™ One-Step RT-PCR. Les valeurs moyennes de Ct ont été analysés
statistiquement. Les valeurs de Ct les plus basses ont été observées en
utilisant le kit Qiagen® RNeasy Mini Kit. Le kit Mag MAX™-96 AI/ND Viral
RNA Isolation Kit en combinaison avec un système automatisé a permis le
traitement d’un plus grand nombre d’échantillons dans une période de temps
plus courte, ce qui serait particulièrement bénéfique lors d’une épidémie de
la maladie de Newcastle. Aucune contamination croisée n’a été observée quel
que soit le kit utilisé.
Mots-clés : ARN, kit, diagnostic, Maladie de Newcastle,
RT-PCR
Introduction
Newcastle disease virus (synonymous with Avian
paramyxovirus 1, NDV), a member of the family
Paramyxoviridae, subfamily Paramyxovirinae, genus
Avulavirus, is a pathogen capable of causing devastating
disease, particularly in poultry [1, 2]. Chickens infected
with NDV may show a wide spectrum of clinical signs that
vary with virus strain, ranging from extremely mild enteric
or respiratory disease (lentogenic strains) to severe systemic
infections with high mortality rates (velogenic strains) [1,
2, 7]. The “gold standard” for identification of the agent
involves isolation and cultivation in embryonated chicken
eggs followed by hemagglutination test, hemagglutination
inhibition test and pathotyping of the virus. Pathogenicity
traditionally is determined by the intracerebral pathogenicity
index. These assays are labor intensive and time-consuming,
requiring up to 10 days to complete [9, 16, 20]. This hinders
the authorities in undertaking adequate measures in a timely
manner to limit the spread and eradicate the infection. The
development and implementation of rapid, reliable, and
high-throughput diagnostic methods for detection of the
virus would provide a valuable contribution to controlling
the disease. The real-time reverse transcriptase-polymerase
chain reaction (rRT-PCR) is a test that satisfies the
requirements for high sensitivity and specificity coupled with
a short turnaround time. [7, 17, 23, 24].
Sensitivity and specificity of rRT-PCR depends on
multiple parameters such as design and concentration of
primers and probes, thermocycler temperature conditions,
and type and concentration of enzymes, presence of
PCR inhibitors etc. [6]. The successful extraction of viral
ribonucleic acid (RNA) is essential for performing rRTPCR [10] and the reproducibility and high sensitivity of
the reaction is dependent upon RNA yield [11, 12]. The
purification of RNA from the other biological substances and
reagent residues is particularly important, as they could exert
an inhibiting effect upon the reaction [8, 11, 13, 18].
There are various techniques for performing RNA
extraction, some using commercial kits, others utilizing
general purpose or “in-house” produced reagents that may
Revue Méd. Vét., 2014, 165, 5-6, 172-175
NDV RNA EXTRACTION – COMPARISON THREE METHODS
vary in their performance. The aim of the this study was to
compare the performance of extraction of NDV RNA with
two commercial kits - Qiagen® Rneasy Mini Kit and Mag
MAX™-96 AI/ND Viral RNA Isolation Kit, and extraction
with TRIZOL® LS, a commercially available RNA extraction
reagent, using rRT-PCR. The length of time to perform the
extraction of a specific number of samples, as well as the
presence of cross contamination was also compared.
Materials and methods
In this study, ten samples selected from proficiency tests
for Avian Paramyxovirus 1 (APMV 1) - APRT Test 07, set
145, sent by the National Veterinary Services LaboratoriesUnited States Department of Agriculture (NVSL-USDA),
Ames, Iowa were used. The samples contained allantoic fluid
from infected embryonated chicken eggs.
Extractions were carried out in triplicate for each sample
using three methods-the Qiagen® RNeasy Mini Kit (Qiagen®,
Valencia, CA, USA), the Mag MAX™-96 AI/ND Viral RNA
Isolation Kit (Ambion®, Austin, TX, USA) and TRIZOL® LS
(Invitrogen, Carlsbad, CA, USA).
All extractions were performed at a temperature of 2022˚С. The extraction with Qiagen® RNeasy Mini Kit was
carried out using a modified method of manufacturer’s
instructions for RNA isolation from animal cells. In
this method 500 µl buffer RLT supplemented with
β-mercaptoethanol (10 µl β-МЕ per 1ml buffer to a final
concentration of 1%) and 250 µl of each sample was used.
The final extraction volume obtained was 50 µl.
The extractions with the Mag MAX™-96 AI/ND Viral
RNA Isolation Kit were carried out using 50 µl of sample, 100
µl of lysis solution, 20 µl of magnetic bead solution (sample
plate), 100 µl of wash solution 1 (1st wash plate), 100 µl of
wash solution 2 (2nd wash plate), 100 µl of wash solution
2 (3rd wash plate), and 50 µl of elution buffer (elution
plate). The extraction was performed in a KingFisher Flex
Automated Purification System (Thermo Scientific, Ontario,
Canada). The program included the following steps: 4 min
lysis and RNA binding to magnetic beads, 1 min washing
with solution 1, two wash steps of 30 s each with solution two
and a 4 min elution step.
The TRIZOL® LS extractions were carried out following
the manufacturer’s instruction for RNA extraction from
biological fluids, i.e. using 250 µl of each sample and 1 ml
TRIZOL® LS. RNA was reconstituted in 50 µl RNase-free
water.
The yields for each of the three methods RNAs were
assayed for the NDV Matrix-Gene by rRT-PCR using the
AgPath-ID™ One-Step RT-PCR (Ambion®, Austin, TX,
USA) and the 7500 Fast Real-Time PCR System (Applied
Biosystems, Foster City, CA). This protocol is approved by
NVSL. The total reaction volume of 25 µl for each sample
consisted of RNase-free water – 0.83 µl; 2х buffer – 12.5 µl;
forward primer - 0.25 µl (20pmol/ µl); reverse primer – 0.25
Revue Méd. Vét., 2014, 165, 5-6, 172-175
173
µl (20pmol/ µl); probe – 0.5 µl (6pmol/ µl); 25x Enzyme Mix
– 1.0 µl; detection enhancer – 1.67 µl and RNA template– 8.0
µl. The primers and probe used in this test were described
previously by Wise et al. [23]. The reaction was carried out in
96-well microplates at the following temperature conditions:
reverse transcription for 10 min at 45˚С; initial denaturation
for 10 min at 95˚С; 40 cycles of denaturation for 10 sec at
94˚С, primer annealing for 30 sec at 56˚С and primer
elongation for 10 sec at 72˚С.
To determine whether the differences of cycle threshold
(Ct) values using the three extraction methods were
significant, the average Ct values were compared using a
One-Way ANOVA test. When significant differences were
identified the Tukey- Kramer’s Multiple Comparison Test
was used to perform non-parametric pair-wise analysis. The
statistical software InStat v. 3.10 (GraphPad Software, La
Jolla, CA, USA) was utilized.
To determine the presence of cross contamination
during the RNA extraction, two negative controls for each
extraction were included, using RNase-free water in place of
the sample. Processing times for the extraction of 30 samples
were measured for each extraction method, assuming the
addition of the first reagent as the start and the elution of
RNA of the last sample as the end of the procedure.
Results
In 7 out of the 10 samples tested with the three RNA
extraction protocols, the M-Gene rRT-PCR specific assay
detected the presence of APMV 1 genome. Negative results
for NDV were recorded for three samples (Numbers 7, 9 and
10) by all three methods, which is in agreement with the
expected results sent by NVSL- USDA (unpublished results).
The obtained Ct values for each of the extraction methods as
well the standard deviation (SD) are shown in Table I. The
differences of mean Ct values for the three RNA extraction
methods were found to be significant by One-Way ANOVA
(P<0,05; n=7). Pair-wise comparisons of Ct values indicated
a significant difference between both the TRIZOL® LS and
the kit Qiagen® RNeasy Mini Kit when compared with the
Mag MAX™-96 AI/ND Viral RNA Isolation Kit (P<0.05,
n=7) but no significant difference between Qiagen® RNeasy
Mini Kit and TRIZOL® LS.
The two negative extraction controls included in each
of the three extraction methods did not produce detectable
fluorescence, as expected. The processing time for 30 samples
with the individual techniques was as follows: Qiagen®
Rneasy Mini Kit – 80 minutes, the operator was occupied
for the full 80 minutes; Mag MAX™-96 AI/ND Viral RNA
Isolation Kit – 40 minutes, with the operator occupied for
20 minutes; TRIZOL® LS – 120 minutes, with the operator
occupied for approximately half this period.
Discussion
There are numerous reports in the literature investigating
and comparing extraction methods for RNA of various
174
DIMITROV (K.) AND COLLABORATORS
Sample number
Extraction technique
Qiagen® Rneasy Mini Kit
TRIZOL® LS
Mag MAX™-96 AI/ND Viral
RNA Isolation Kit
1
18.76±0.22
18.95±0.26
22.90±0.26
2
24.22±0.31
25.41±0.49
27.54±0.39
3
22.04±0.20
22.64±0.17
26.07±0.24
4
21.56±0.32
22.46±0.23
25.89±0.34
5
24.76±0.29
26.02±0.22
31.89±0.34
6
24.91±0.37
26.01±0.20
29.48±0.37
7
neg
neg
neg
8
28.02±0.39
27.36±0.10
30.67±0.41
9
neg
neg
neg
10
neg
neg
neg
еxtraction controlb 1
neg
neg
neg
еxtraction control 2
neg
neg
neg
a
a neg = negative
b extraction control = RNase-free water in place of sample
Тable I: Cycle threshold (Ct) values (mean ± SD) obtained for RNA extracted by three different techniques. All extractions were conducted in triplicate and
the yields for each of the three methods RNAs were assayed for the NDV Matrix-Gene by rRT-PCR (AgPath-ID™ One-Step RT-PCR).
disease agents using various sample matrices [3, 4, 13, 15,
17, 19, 21, 22, 24], but data referring to APMV 1 is scarce.
The results of the present study (Table I) and the statistical
analysis of data showed lower Ct values of rRT-PCR following
extraction with either Qiagen® RNeasy Mini Kit or Trizol
LS vs. Mag MAX™-96 AI/ND Viral RNA Isolation Kit. The
results demonstrated no statistically significant difference in
sensitivity between extractions with Qiagen® RNeasy Mini
Kit and TRIZOL® LS. In contrast to this study, Crossley et
al. [5] observed a higher sensitivity for the magnetic bead
method when comparing extraction of NDV RNA using
TRIZOL® LS with a commercial kit using magnetic bead
technology (Ambion, Austin, TX. USA). It is important to
note that the authors used field samples in their experiment.
Such samples could contain different biological substances in
comparison to the samples used in the present study and this
could influence the RNA purification process. The extraction
system used by Crossley et al. [5] was not fully automated,
but used magnetic stands with manual washing. Marshall
and Bruggink [14] concluded that some automatic nucleic
acid extraction methods may result in reduction of detection
sensitivity.
The initial amount of the sample in each extraction
protocol could also be important. The sample volume
used in extraction with Mag MAX™-96 AI/ND Viral RNA
Isolation Kit was five times lower as compared to the other
two methods. However, the aim of the present study was to
compare the extraction methods as recommended by the
manufacturers.
The negative results of the extraction controls and
of samples Numbers 7, 9 and 10 confirm lack of cross
contamination in all three investigated RNA isolation
techniques. This corresponds to data reported in similar
studies [13, 17, 21]. However, procedures using Qiagen®
Rneasy Mini Kit and TRIZOL® LS are manual and cross
contamination would depend on the skills of the person
carrying out the extractions.
In conclusion, of all three tested methods for extraction
of RNA from NDV, the lowest Ct values obtained with
rRT-PCR were observed when using the Qiagen® Rneasy
Mini Kit. The Mag MAX™-96 AI/ND Viral RNA Isolation
Kit in combination with an automated system allowed the
processing of a greater number of samples in a shorter
period of time (one-half to one-third compared to the two
other techniques), this would be particularly useful in cases
of the high volume of surveillance sample testing required
following a Newcastle disease outbreak.
Acknowledgments
All tests used in this study were carried out in the
Texas Veterinary Medical Diagnostic Laboratory, College
Station, TX, USA and were supported by the Norman E.
Borlaug International Agricultural Science and Technology
Fellowship Program. The authors wish to thank Celia
Abolnik and Wendy Shell for their useful comments on the
manuscript.
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