Point of Care Strategy for Rapid Diagnosis of Novel A/
H1N1 Influenza Virus
Antoine Nougairede1,2, Laetitia Ninove1,2, Christine Zandotti1, Xavier de Lamballerie1,2, Celine Gazin1,
Michel Drancourt1,3, Bernard La Scola1,3, Didier Raoult1,3, Remi N. Charrel1,2*
1Fe ´de ´ration de Microbiologie, Assistance Publique - Ho ˆpitaux de Marseille, Marseille, France, 2Unite ´ des Virus Emergents, UMR 190 ‘‘Emergence des pathologies virales’’,
Universite ´ de la Me ´diterrane ´e & Institut de Recherche pour le De ´veloppement, Marseille, France, 3Unite ´ de Recherche sur les Maladies Infectieuses et Tropicales
Emergentes UMR CNRS 6236 IRD 3R198, IFR 48, Faculte ´ de Me ´decine, Universite ´ de la Me ´diterrane ´e, Marseille, France
Background: Within months of the emergence of the novel A/H1N1 pandemic influenza virus (nA/H1N1v), systematic
screening for the surveillance of the pandemic was abandoned in France and in some other countries. At the end of June
2009, we implemented, for the public hospitals of Marseille, a Point Of Care (POC) strategy for rapid diagnosis of the novel
A/H1N1 influenza virus, in order to maintain local surveillance and to evaluate locally the kinetics of the pandemic.
Methodology/Principal Findings: Two POC laboratories, located in strategic places, were organized to receive and test
samples 24 h/24. POC strategy consisted of receiving and processing naso-pharyngeal specimens in preparation for the
rapid influenza diagnostic test (RIDT) and real-time RT-PCR assay (rtRT-PCR). This strategy had the theoretical capacity of
processing up to 36 samples per 24 h. When the flow of samples was too high, the rtRT-PCR test was abandoned in the POC
laboratories and transferred to the core virology laboratory. Confirmatory diagnosis was performed in the core virology
laboratory twice a day using two distinct rtRT-PCR techniques that detect either influenza A virus or nA/N1N1v. Over a
period of three months, 1974 samples were received in the POC laboratories, of which 111 were positive for nA/H1N1v.
Specificity and sensitivity of RIDT were 100%, and 57.7% respectively. Positive results obtained using RIDT were transmitted
to clinical practitioners in less than 2 hours. POC processed rtRT-PCR results were available within 7 hours, and rtRT-PCR
confirmation within 24 hours.
Conclusions/Significance: The POC strategy is of benefit, in all cases (with or without rtRT-PCR assay), because it provides
continuous reception/processing of samples and reduction of the time to provide consolidated results to the clinical
practitioners. We believe that implementation of the POC strategy for the largest number of suspect cases may improve the
quality of patient care and our knowledge of the epidemiology of the pandemic.
Citation: Nougairede A, Ninove L, Zandotti C, de Lamballerie X, Gazin C, et al. (2010) Point of Care Strategy for Rapid Diagnosis of Novel A/H1N1 Influenza
Virus. PLoS ONE 5(2): e9215. doi:10.1371/journal.pone.0009215
Editor: Robyn Klein, Washington University School of Medicine, United States of America
Received November 21, 2009; Accepted January 25, 2010; Published February 17, 2010
Copyright: ? 2010 Nougairede et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was partly funded by AP-HM Public Hospital system and by the FP7 European project nu228292 European Virus Archive (EVA). The funders
had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
In late April 2009, The World Health Organization (WHO)
announced the emergence of a novel A/H1N1 influenza virus
(nA/H1N1v). This virus spread rapidly, and after two months the
WHO raised the alert level from phase 5 to phase 6 defining the
first influenza pandemic of the 21st century . At the beginning
of the pandemic, some countries established measures to identify
all possible cases, but rapidly and due to the constant increase of
suspected cases, they decided to abandon the systematic screening.
In France, the initial strategy which commenced at the end of
April 2009 relied on the early identification of suspect cases which
were directed into the hospital system to be tested for nA/H1N1v,
the positive cases being further hospitalized in an isolation ward to
reduce secondary transmission. This approach provided informa-
tion concerning the kinetics of the pandemic when the most
positive cases were acquired abroad. The systematic screening of
all suspect cases was abandoned on July 7thand replaced by
sentinel systems to estimate the number of cases from surveillance
of certain populations and to target groups with higher risk of
morbidity or mortality . This cessation of laboratory-confirmed
cases eliminated the possibility of a reliable estimation of the
evolution of the pandemic in France. Figures varying from 28,000
(Groupes Re ´gionaux d’Observation de la Grippe, France) to
130,000 (Re ´seau sentinelles, France) cases weekly were claimed
during the same period depending on the source data . The
data gathered at the beginning of the pandemic in Europe clearly
indicate that the positive predictive value of the clinical
examination during examination by general practitioners or
infectious disease specialists ranged between 8 and 25%, which
is very low, thus justifying a laboratory-based diagnostic approach
[2,4,5,6]. This is a strong argument to maintain laboratory
confirmation of nA/H1N1v suspect cases to provide robust
epidemiological data. Even if the results are biased, the calculated
proportion of laboratory-confirmed nA/H1N1v infection cases
amongst suspect cases enables extrapolation to provide a
PLoS ONE | www.plosone.org1February 2010 | Volume 5 | Issue 2 | e9215
reasonable estimation of the total number of cases. Because of the
innate evolving nature of an epidemic/pandemic caused by a
transmissible agent, such an extrapolation must be periodically
updated for comparison with the kinetics of the pandemic from
unambiguous data as derived from laboratory-confirmed diagnos-
To maintain systematic surveillance in the public hospitals of
Marseille, we organized our two POC laboratories to perform
rapid diagnosis of nA/H1N1v. Here we describe the POC
organization, the results of POC laboratories during three months,
and the sensitivity and the specificity of the POC-based results
during this period.
Materials and Methods
Following national regulations under the term of Biomedical
Research (Loi Huriet-Se ´rusclat (loi 881138)), the signature at the
hospital entrance office warrants that all samples done during
hospitalization for diagnostic purpose are accessible for research
(excluding human genetic research) without specific consent of the
patient and then ethics approval was not asked for regarding the
terms of the Loi Huriet-Se ´rusclat (loi 881138).
For each patient, specimens from both nostrils were obtained
with the same Virocult swab (Virocult MW950; Medical Wive and
Equipment Co.). Specimens collected were rapidly transported at
room temperature for testing in the POC laboratory.
Processing, Aliquoting, Internal Control Spiking and
Rapid Influenza Diagnostic Test (RIDT)
In a class-2 biosafety cabinet, nasal swabs were resuspended in
1 mL of sterile PBS solution and transferred into a 2 mL cryotube.
Two aliquots were prepared respectively for RIDT and nucleic
acid extraction. The remaining material, to be tested in the core
laboratory was (i) either stored at 280uC in the La Timone
hospital POC laboratory, (ii) or stored at 220uC temporarily in
the North hospital POC laboratory and shipped to La Timone
core laboratory on dry ice every 3 hours. RIDT was performed
using the Directigen EZ influenza A+B test (BD EZ Flu A+B,
Becton, Dickinson and Company) according to the manufacturer’s
One 200-mL aliquot of suspended nasal swab was added to a
2 mL tube containing 200 ml of AVL buffer and 10 ml of MS2
phage internal control and incubated for 10 minutes at room
temperature for inactivation. Extracted nucleic acids were then
purified using the EZ1 Virus Mini Kit v2.0 (elution volume: 90 ml)
onto the EZ1 Biorobot (both from Qiagen).
Quantitative Real Time PCR Assays
Each sample was tested by two rtRT-PCR systems: (i) a rtRT-
PCR assay using SYBR Green technology targeting all influenza A
viruses , and (ii) a rtRT-PCR assay specific for the nA/H1N1v.
The latter was developed and provided by the French Influenza
Reference National Center . This test was used in both POC
laboratories on a two-block SmartCycler system (reaction volume:
25 ml), and in the core laboratory on either an Mx3005p
thermocycler (Stratagene) or an LC480 thermocycler (Roche)
(reaction volume: 50 ml). rtRT-PCR reactions were performed
using the SuperScript III Platinum One-Step qRT-PCR kit
(Invitrogen) under the same conditions in POC and core
laboratories. Standard quantities of 2X PCR Master Mix and
SuperScript III RT/Platinum Taq Mix were added to the mixture
reaction containing 0.8 mM of each primers (GRswH1-349Fw
GAGCTAAGAGAGCAATTGA; GRswH1-601Rv GTAGATG-
GATGGTGAATG) and 0.2 mM of the probe (GRswH1-538
Probe TTGCTGAGCTTTGGGTATGA Fam BHQ-1).
A volume of 5 ml and 10 ml of total RNA was used for 25 ml and
50 ml final reaction volumes respectively. Positive controls
consisted of (i) an in-vitro synthesized RNA transcript, and (ii) a
patient-derived positive sample that was used in the core
laboratory only. Reaction conditions were as follows: reverse
transcription at 50uC for 15 min, initial denaturation at 95uC for
2 min, followed by 45 cycles of 95uC for 15 s, 60uC for 40 s.
POC Laboratory Organization in Marseille
In Marseille, the recent reorganization of the health structures
(4 public hospitals) was achieved through the implementation of
unique core laboratory for Clinical Microbiology. Centralization
of the laboratories so that they serve several hospitals maximizes
the efficiency of testing, thus reducing costs but has the
disadvantage of poor communication with physicians in clinical
wards and problems of specimen transfer . As countermeasures,
we decided to establish two Point Of Care (POC) laboratories
located in the vicinity of the emergency units (see Figure 1) to
reduce delays due to sample transportation and to reduce time to
obtain the results for selected analyses. Open 24 h/24 and
operated by one person, POC laboratories can rapidly perform a
large panel of analyses (see Table 1). For selected parameters, the
results were confirmed by the core virology laboratory.
POC Laboratory Adaptation for Diagnosis of nA/H1N1v
When secondary cases of nA/H1N1v were documented in
France, we decided to implement rapid detection of nA/H1N1v in
the POC laboratories to provide rapid diagnosis and to study levels
and variation of virus circulation. Both POC laboratories were
adapted to process clinical specimens under appropriate biosafety
conditions, and to perform nA/H1N1v detection via rapid
Figure 1. POC laboratories organization in Marseille.
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influenza diagnostic tests (RIDT), and real time RT-PCR (rtRT-
PCR) on SmartCycler (Cepheid). POC operators were trained to
work in class II laminar hoods, and to perform RIDT and rtRT-
PCR for influenza virus. The rtRT-PCR detection was performed
using the test recommended by the French Influenza Reference
National Centre which was adapted on SmartCycler . When
received at the POC laboratory, specimens were systematically
spiked with an internal control that monitored all steps of the
technical process from nucleic acid extraction to rtRT-PCR.
Comparative evaluation with the same protocol operated in an
MX3005P thermocycler (Stratagene) showed a lower sensitivity for
low copy number specimens, but no significant difference in term
of specificity (data not shown). Therefore positive rtRT-PCR
results were validated at the POC level. The training duration for
all steps of sample analysis in POC was shorter than one working
day (see table 2). The cost necessary to implement POC capacity
to perform nucleic acid extraction and rtRT-PCR assay was
estimated at 110,000 J for the equipment and 19 J per sample
(see table 2). The maximal flow capacity (upper limit) of the POC
laboratory for detection of nA/H1N1v was theoretically 36
samples per day (see figure 2). In our experience, a POC
laboratory was saturated when more than 20–25 samples to be
tested for nA/H1N1v were received per day. This discrepancy is
mainly due to two points: (i) samples were not received
Table 1. List of POC analysis.
Pathogen agent Specimen Test/TargetTime for result
Pharyngitis Group A Streptococcuspharyngeal swabICT/Antigen30 min
Epstein Barr Virusserum ICT/Antibody30 min
Pneumopathy Mycoplasma pneumoniaesputum or nasopharyngeal aspirateqPCR/Genome 3 h30
Bordetella pertussis sputum or nasopharyngeal aspirate qPCR/Genome3 h30
Legionella pneumophilaurine ICT/Antigen30 min
Influenza A/B virus nasopharyngeal aspirate ICT/Antigen30 min
Respiratory syncytial virusnasopharyngeal aspirate ICT/Antigen30 min
DiarrhoeaRotavirus and Adenovirus stool ICT/Antigen30 min
Clostridium difficilestoolICT/Antigen 30 min
MeningitisNeisseria meningitidis Cerebrospinal fluidqPCR/Genome 3 h30
Streptococcus pneumoniaeCerebrospinal fluid qPCR/Genome 3 h30
Mycoplasma pneumoniaeCerebrospinal fluidqPCR/Genome 3 h30
Herpes Simplex Virus 1/2 Cerebrospinal fluid qPCR/Genome3 h30
Enterovirus Cerebrospinal fluidqRT-PCR/genome 2 h30
Cryptococcus Cerebrospinal fluidICT/Antigen 45 min
Gynaecology Group B Streptococcusvaginal swab qPCR/Genome2 h30
HIV 1/2serum ICT/Antibody30 min
Tropical fever Plasmodiumblood ICT/Antigen30 min
Dengue VirusserumICT/Antigen and Antibody 30 min
Blood exposure accident HIV 1/2serumICT/Antibody 30 min
Risk of Tetanus TetanusserumICT/Antibody 30 min
GastroenterologyHelicobacter pyloriurine ICT/Antigen30 min
Urinary tract infectionUrinary reactive stripstool colorimetric assay30 min
Procalcitonine Procalcitonineserum ICT/Antigen45 min
ICT: immuno-chromatographic test.
Table 2. Equipment, cost and training duration required for POC laboratory implementation.
Maximum number of
specimens tested per run
RIDT influenza A+B (Directgen Influenza Test Kit, Becton Dickinson)8 J/sample-30 min
Extraction Instrument (EZ1 BioRobot, Qiagen)30 000 J
6 (45 min) 1 h
Viral RNA extraction (EZ1 Virus Mini Kit v2.0, Qiagen)5 J/sample
rtRT-PCR for nA/H1N1v and internal control (SSIII platinum one step qRT-PCR, Invitrogen)6 J/sample14 (2 h30)3 h
Thermocycler (Smart cyler, Cepheid) two blocks75 000 J
<5 000 J
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continuously during the 24hperiod, (ii) POC diagnostic activities
were maintained and operators had to process other samples for
detection of viruses and bacteria other than nA/H1N1v (see
POC Laboratory Data from June 23rdto September 27th
From June 23rd2009 to September 27th2009, 1974 samples
were analyzed in the two POC laboratories (see figure 3). The
majority of samples originated from emergency wards (pediatrics
and adults) (64.7%) and from the specific influenza consultation
located in the North Hospital (9.5%). The median and mean ages
were 21 years and 25.4 years respectively for these 1974 patients
(see table 3). The two POC laboratories processed a similar
number of specimens. At the beginning of the period, each
laboratory tested about 30–40 samples per week. Then there was
an obvious increase in the activity with more than 150 samples
weekly from mid August to Sept 7th, and more than 250 samples
weekly for the three last weeks of September (see figure 3). In late
August, this rapid increase of clinical specimens constrained us to
stop performing rtRT-PCR, while sample processing, RIDT and
nucleic acid extraction were maintained at the POC level.
From late June to late August 990 samples were processed in
POC laboratories. During this period, a proportion of samples, for
which nucleic acid extraction was performed at POC level, was
rtRT-PCR tested only in the core laboratory (427 samples); 563/
990 samples were tested using the rtRT-PCR on SmartCycler in
POC laboratories, of which 33 (5.9%) were positive. From late
August to late September, all samples extracted by POC were
tested directly by the core laboratory twice a day.
Among the 1974 samples received in POC laboratories, 111
(5.6%) samples were positive after core laboratory confirmation
using rtRT-PCR. A total of 1974 RIDT were performed, of which
64 were positive. These 64 RIDT positive samples were confirmed
by A/H1N1 rtRT-PCR, then RIDT specificity was 100% (no false
positive). Of the 111 core laboratory positive samples, 47 were not
detected using RIDT (false negative). Therefore, the sensitivity of
the POC strategy for A/H1N1 was 57.7% using RIDT and 84.6%
using POC rtRT-PCR assay. We determined the positive and
negative predictive value (PPV and NPV) for each step of the POC
process (see figure 4).
From mid-August, the number of samples received for testing
started to increase. In parallel, the number of positive samples
equal to or higher than 10 per week was observed (except for week
36; see figure 3). During September, we observed an increase of
specimens received for testing, whereas the number of positive
samples remained stable being constantly lower than 6% of tested
samples (see figure 3).
The delay necessary to obtain the results was (i) ,2 h for the 64
samples with positive RIDT and (ii) 4–7 h for the 546 samples -
Figure 2. Flow capacity in POC laboratory for detection of nA/
H1N1v. Black boxes represent sample receipt, registration, processing,
aliquoting, internal control spiking and rapid influenza A+B test (2 h for
6 samples). Light grey boxes represent RNA extraction (6 maximum)
and PCR-mix preparation (1 h30). Dark grey boxes represent rtRT-PCR
(detection of nA/H1N1v on SmartCycler), interpretation and results
validation (3 h).
Figure 3. Time distribution of samples tested and positive for nA/H1N1v in POC laboratories. Positive samples correspond to rtRT-PCR
core laboratory-confirmed results regardless the result obtained at the POC level.
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with negative RIDT - tested using rtRT-PCR in POC laborato-
ries. For the remaining 1364 samples, the uninterrupted nucleic
acid extraction at POC level reduced the time to obtain the results
(10–24 h). Computer-based communication of results also saved
the time that would normally be dedicated to telephone calls for
both the physician and the POC operator.
To assess circulation of nA/H1N1v in infants and in adults, we
compared the percentage of positive samples in pediatric and adult
emergency wards (Table 3). From late June to mid-August,
pediatric and adult emergency wards tested about 3 patients per
day. The number of positive samples was low until August, then it
sharply increased. In August and September, the percentage of
positive samples progressively increased for children and decreased
for adults, indicating a tendency that persisted in October (data
not shown; figure 5).
Core Laboratory Data from April 25thto September 27th
The core laboratory processed confirmatory tests for all samples
received at POC laboratories using (i) the rtRT-PCR nA/H1N1v
assay aforementioned and (ii) an rtRT-PCR SYBR Green system
which detected all Influenza A viruses . Both PCR reactions
were performed on either the Mx3005p thermocycler (Stratagene)
or the LC480 thermocycler (Roche). From April 25th2009 to
September 27th2009, the core laboratory received and processed
3609 samples for nA/H1N1v detection. A total of 339 samples
were positive (9.4%). During the entire period, our laboratory was
in charge of testing human samples corresponding to 9 French
departments (south-eastern France) representing 8 million inhab-
itants. From late April to early July, the authorities recommended
to test all suspect cases . During this period up to 50 specimens
were received weekly. On June 23rd2009, we started to implement
the POC strategy for all samples received from Marseille public
hospitals, while samples received from hospitals and practitioners
outside of Marseille were processed directly in the core laboratory.
This strategic change was synchronous with a drastic increase in
the weekly number of samples. During this period, about 100–150
samples were received each week until mid-August which
witnessed an obvious increase in the activity with more than 200
samples during the next three weeks and more than 500 samples in
the following three weeks of September (see figure 6).
POC strategy for nA/H1N1v requires minimal training,
necessitates a small dedicated laboratory area available in any
hospital setting and can be operated by one person. It enables the
hospital to provide diagnosis within the period of clinical illness.
This is imperative if one wishes to take the decision to treat the
patient, to decide to isolate the patient to prevent nosocomial
transmission, or to discharge the patient. It provides reliable data
for studies of the local epidemiology and its evolution. It also
contributes to defining and monitoring the epidemiology on a
larger scale (regional, national, international), via extrapolation
based on the percentage of laboratory-confirmed cases compared
with suspect cases. The usefulness of the POC strategy in the
context of the nA/H1N1v pandemic demonstrates that POC
could usefully be implemented throughout the country not only for
emergency situations but also as a daily tool to improve the quality
of care for hospitalized patients, by shortening the delays and
allowing decisions during the period of clinical presentation. It is
Table 3. Characteristics and results for each group described in this study.
Samples positive for
seasonal influenza virus
All POC samples25.421 1974111 (5.6%)9
Adults emergency42.036 59628 (4.7%)2
Pediatrics emergency 4.03 68245 (6.6%)1
Figure 4. Flow chart of POC samples with sensitivity and specificity of each step. PPV: Positive predictive value. NPV: Negative predictive
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likely that hospitalization costs would also directly benefit from
POC strategy, through reduction of the duration of hospitalization
as previously demonstrated [9,10]. Our experience demonstrates
that POC laboratory implementation reduces the time necessary
to obtain the results in all cases, even when the molecular
techniques are not performed at the POC level. Although, the
sensitivity of POC tests is usually lower than that of rtRT-PCR
techniques, their high PPV enables clinical decisions to be taken
much more rapidly than in the standard diagnostic approach.
One can argue that an efficient transportation system may be
less expensive than setting up a POC facility because of the short
distance (less than 10 km) between the North Hospital and the
core laboratory. However, our experience is that delays due to
transfer for medical ward to core laboratory can vary greatly (up to
7 hours). In our hospital system, samples transportation is
operated by messengers (with cars). The long delays may be
sometimes due to heavy traffic between the two hospitals (more
than 1 hour). Beside, as MDs, we have no hierarchical authority
on the messengers. This situation has been discussed with the
administrative head of the hospital, but no satisfactory solution
could be found. This is the reason why the POC laboratory
solution has been considered and developed.
In our opinion, the decision to abandon systematic laboratory
testing of suspect patients was equivalent to breaking the
thermometer while attempting to define the body temperature
curve. We believe that the application of diagnostic tools for
respiratory tract infections (RTI) enables the doctors and nurses to
work efficiently to combat this group of diseases that the most
common cause of death worldwide  and the most neglected
cause of reduced longevity . In the current pandemic situation
where less than 10% of tested specimens were found positive for
nA/H1N1v, the economic impact of rapid testing through the
POC strategy is very important. Indeed, all negative patients - the
large majority of suspect patients for the considered study period -
can therefore return to their professional activities immediately.
The re-admission of students in the educational course is often
conditioned by a certificate assessing the absence of contagiousness
for nA/H1N1v, which needs to be based on specific virologic
diagnostic tests such as those implemented in the POC strategy.
Finally, a better knowledge of the local and seasonal ecology of
RTI microorganisms must help to determine the panel of agents to
be tested at the POC level. As an example, the findings that 34%
of suspected nA/H1N1v infections were in fact due to rhinoviruses
 should theoretically justify POC testing for these viruses. It is
Figure 5. Time distribution of samples received/positive from adult and pediatric emergency wards. Positive samples correspond to
rtRT-PCR core laboratory-confirmed results regardless the result obtained at the POC level. w: p,0.01 (chi-square test).
Figure 6. Number of samples tested/positive in the core laboratory from April to September 2009. Samples were tested using two rtRT-
PCR assays (see materials and methods).
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also likely that the panel of POC detected pathogens will Download full-text
progressively increase to other agents causing respiratory tract
infections. To address influenza morbidity and mortality, the
approach based on systematic screening is more efficient than
passive methods that produce underestimated data, in part due to
the fact that they cannot consider the role of unrecognized
influenza infection as a decisive co-morbidity factor in patients
with underlying cardiovascular disease, hypertension, chronic
pulmonary diseases and endocrine disorders . Therefore
passive surveillance tends to hinder the knowledge of epidemiology
of this pandemic . Systematic detection for each patient with
severe influenza-associated pathology like acute respiratory distress
syndrome  or for each patient with high mortality risk like
pregnant woman  may contribute to appreciating the true
incidence of influenza infection in such specific groups.
The authors wish to thank all the interns involved in POC activities from
May to October 2009, the technicians of the Virology laboratory and
BIOTOX unit, and Professor Ernest A. Gould for English improvement.
Conceived and designed the experiments: AN XdL MD BLS DR RNC.
Performed the experiments: AN LN CZ CG. Analyzed the data: AN LN
CZ XdL CG MD BLS DR RNC. Contributed reagents/materials/analysis
tools: AN LN CZ CG. Wrote the paper: AN DR RNC.
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