Development of an on-site diagnostic system for infectious diseases:
evaluation using blood samples containing the bovine leukemia DNA
Yasuko Yamazaki1, Uraiwan Seo1,
Konomu Nagao2, Hirohisa Mekata3,
Wataru Yamazaki1*
1Center for Southeast Asian Studies, Kyoto University,
46 Shimoadachicho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan.
2Department of Veterinary Science, Faculty of
Agriculture, University of Miyazaki, 1-1 Gakuen Kibanadai-nishi,
Miyazaki, Miyazaki, 889-2192 Japan.
3Organization for Promotion of Tenure Track,
University of Miyazaki, Miyazaki 889-2192, Japan
Running head: On-site diagnosis in epidemic field settings
*Corresponding author:
Wataru Yamazaki, Center for Southeast Asian Studies, Kyoto University,
46 Shimoadachicho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
Tel.: +81 (0) 75-753-9618; Fax: +81 (0) 75-761-2701
E-mail: yamazaki@cseas.kyoto-u.ac.jp
Summary
Along with progress in globalization of society, the spread of
infectious diseases has accelerated worldwide. Although the deployment
of highly sensitive genetic tests is essential for early diagnosis and
early containment of potential epidemics, tedious and expensive nucleic
acid extraction steps represent a major drawback. Here we developed a
simple and rapid DNA extraction method, named as an EZ-Fast kit,
applicable to the epidemic field setting. The kit does not require
advanced laboratory equipment or expensive DNA extraction kits and
achieves crude DNA extraction within 10 min at extremely low cost. When
combined with real-time PCR and LAMP analyses, the performance of the
method, using 183 bovine blood samples, was similar to that of the
existing DNA extraction method: 92.5% (135/146) (real-time PCR) and
93.7% (133/142) (LAMP) diagnostic sensitivities, and 100% diagnostic
specificities. The developed system provides a powerful tool to
facilitate on-site diagnosis in an epidemic field setting.
Keywords: EZ-Fast; Field setting; LAMP; On-site; Real-time PCR
Introduction
With progress in globalization, the risk of spreading human and animal
infectious diseases has accelerated worldwide (Howson et al, 2018; Huang
et al, 2020; OIE, 2018b; WHO, 2020; Yamazaki et al, 2019). Pandemic
spread of human pathogens such as SARS-CoV-2 (COVID-19) can cause
numerous fatalities worldwide (Huang et al, 2020; WHO, 2020). Further,
transboundary animal diseases cause serious global economic damage to
livestock and meat industries worldwide (Howson et al, 2018; OIE, 2018b;
Yamazaki et al, 2013, 2019). It is therefore important to minimize the
spread of pathogens through correct and rapid diagnosis of diseases of
humans and animals. This in turn highlights the necessity for on-site
diagnosis in epidemic field settings (Almassian, et al, 2013; Armson et
al, 2017; Howson et al, 2018; Madi et al, 2012).
The immunochromatographic assay (ICA) is widely used for rapid screening
on farms (OIE, 2018b). Although ICA is useful for rapid diagnosis of
infected animals with high pathogen levels, its lack of sensitivity is a
major drawback, because detection using ICA requires
≥105–106 infectious units per 1 g
or 1 ml of sample, potentially leading to false-negative diagnoses of
recently-infected animals with a low number of pathogens (OIE, 2018b).
Deployment of genetic tests for on-site diagnosis in epidemic field
settings is an attractive option to achieve early containment because of
their high diagnostic performance (Almassian, et al, 2013; Kurosaki et
al, 2016; Howson et al, 2018). For example, several studies achieved
expedited on-site diagnosis in epidemic field settings of humans and
animals through the use of portable, battery-powered real-time PCR and
loop-mediated isothermal amplification (LAMP) devices (Armson et al,
2017; Howson et al, 2018; Kurosaki, et al, 2016; Madi et al, 2012). The
nucleic acid extraction step requires expensive commercial extraction
kits, however, and is therefore a major disadvantage. Therefore, a
simple dilution technique with nuclease-free water was applied to
successfully detect target pathogens (Howson et al, 2018; Waters et al,
2014). However, this simple technique decreases analytical sensitivity
(limit of detection [LOD]) in the range of a
101–103-fold reduction compared
with conventional laboratory nucleic acid extraction methods (Howson et
al, 2018). More sophisticated approaches that minimize false-negative
results will enable simple and sensitive diagnosis.
Sodium dodecyl benzenesulfonate (SDBS) is an attractive candidate to
develop a simple, rapid, and cost-effective nucleic acid extraction
method using real-time PCR and LAMP amplification (Sekikawa, 2017). SDBS
is an analog of sodium dodecyl sulfate (SDS), which provides equivalent
performance for DNA extraction from cells with lower inhibitory effects
than SDS (Sekikawa, 2017). Here, we developed a simple and
cost-effective on-site diagnosis system through the combination of rapid
DNA extraction using SDBS, named as an EZ-Fast kit, and real-time PCR
and LAMP amplifications that maintains diagnostic accuracy. For this
purpose, we used 183 bovine blood samples containing bovine leukemia
virus (BLV) to evaluate diagnostic performance compared with a
conventional automated DNA extraction method.
2. Materials and Methods
The EZ-Fast kit for on-site diagnosis was developed by combining a
portable heat-block and a dry-cell battery-powered tabletop centrifuge,
which achieves simple DNA extraction using SDBS. Unpurified DNA is
subsequently amplified using a real-time PCR and a portable
battery-powered LAMP device (Fig. 1). To evaluate the performance of
this kit, we compared results with those of the gold standard
(conventional method) real-time PCR and LAMP analyses of DNA using an
automatic nucleic acid extractor.
2-1. Bovine blood samples.
We used 183 bovine blood samples obtained from cattle farms in
Kagoshima, Miyazaki, and Oita prefectures in Japan from March 2017 to
January 2020. EDTA was added to these samples, which were collected at
the farms, kept cool, and delivered to our laboratory where they were
stored 4 °C. DNA was immediately extracted from the remaining bovine
blood samples and used for real-time PCR and LAMP analyses; otherwise,
they were stored at –80 °C.
2-2. DNA extraction using the EZ-Fast kit.
For on-site diagnosis in epidemic field setting, each crude DNA sample
was prepared as follows: 0.5 ml of bovine blood was added to a 1.5-ml
microcentrifuge tube, and 0.5 ml of SDBS (Tokyo Chemical Industry Co.,
Tokyo, Japan) was added and mixed by pipetting 5–10 times. The sample
tube was placed in a portable heat block (Mini heating dry bath
incubator, MD-MINI, Major Science, Co. Ltd., Saratoga, CA, USA),
incubated at 95 °C for 5 min, and centrifuged in a dry-cell
battery-powered tabletop centrifuge (Puchimaru 8, Wakenbtech, Co., Ltd.,
Kyoto, Japan) for 5 min at full speed (6,000 rpm, 1,260–2,840 xg ) at room temperature. A 20-µl sample of the supernatant was
transferred to a new 0.2-ml microcentrifuge tube for real-time PCR and
LAMP analyses. Special attention was payed to retrieving the very top
layer of the supernatant to avoid contamination with solid blood debris
that may contain DNA polymerase inhibitors. The protocol for DNA
amplification steps is shown in Fig. 1. Each 200-μl blood sample was
then added to a 1.5-ml screw-capped tubes and added to 50 μl of
RNase-free water using the magLEAD 6gC automated extraction platform
(Precision System Science Co., Matsudo, Japan) for conventional
real-time PCR and LAMP analyses.
2-3. Real-time PCR.
Using a LightCycler 480 System II (Roche Molecular Systems, Pleasanton,
CA, USA) or a QuantStudio 3 (Thermo Fisher Scientific, Inc., Waltham,
MA, USA), real-time PCR was performed according to the World
Organization for Animal Health (OIE) Manual of Diagnostic Tests and
Vaccines for Terrestrial Animals (2018a) based on pol sequences
reported by Rola-Łuszczak, and colleagues (2013). Reactions were
slightly modified using shorter times for denaturation and annealing
according to the instructions provided with the probe qPCR mix (TaKaRa
Bio Inc., Otsu, Japan). Briefly, 20 µl of rPCR reaction mixtures
comprising 10 µl of 2x probe qPCR mix (TaKaRa Bio), 0.08 µl of each
primer (100 pmol/µl; Hokkaido System Science, Sapporo, Japan), 0.04 µl
of probe (100 pmol/µl; Hokkaido System Science), 7.8 µl of nuclease-free
water (TaKaRa Bio), and 2 µl of the DNA template. The cycling conditions
were as follows: one cycle at 95 °C for 20 s, 45 cycles each at 95 °C
for 5 s, and 60 °C for 30 s. The threshold cycle (CT)
value used as the cut-off was 40.95 according to the OIE Manual (2018a).
2-4. LAMP.
LAMP was performed using a Genie III (OptiGene, Horsham, UK) according
to the LTR sequences reported by Komiyama, et al. (2009) with a slight
modification as follows: Amplification was performed at 63 °C for 30
min, followed by inactivation at 98 °C for 2 min, and cooling to 80 °C
for annealing, with ramping at 0.05 °C/s. The 20-µl LAMP reaction
comprised 10 µl of a Loopamp DNA amplification kit (Eiken Chemical, Co.,
Ltd., Tokyo, Japan) or an in-house LAMP reaction mixture with the
identical composition (Yashiki et. al., 2019); 0.32 µl each of FIP and
BIP primers (100 pmol/µl); 0.16 µl each of LF and LB primers (100
pmol/µl); 0.04 µl each of F3 and B3 primers (100 pmol/µl); 0.8 µl ofBst polymerase 2.0; 0.8 µl of CFI (Colori- Fluorescent Indicator,
comprising 967 µl of distilled water, 30 µl of 100 mmol/µl
hydroxynaphthol blue [MP Biomedicals, Aurora, OH, USA], and 3 µl of
Gel Green [x10 000; Biotium, Hayward, CA, USA]); 5.36 µl of
nuclease-free water; and 2 µl of the DNA template. CFI was added to
accelerate the amplification rate and enable analysis of fluorescence
(Hayashida et., al., 2015; Yashiki et. al., 2019). LAMP primers were
produced by Hokkaido System Science (Sapporo, Japan) (column-grade
purification). The LAMP results were interpreted as positive when the
fluorescence intensity reached 10,000 within 30 min of amplification,
and the annealing temperature (Ta) ranged between 81.5 °C and 84.5 °C.
Time-of-positivity (Tp) was automatically calculated using the Genie III
setting. Details of the primers and probes are shown in Table 1
(Rola-Łuszczak et al, 2013; OIE, 2018; Komiyama et al, 2009).
2-5. LOD
The LOD was determined using 10-fold serial dilutions of BLV
DNA-positive blood samples. We prepared five randomly chosen pooled BLV
DNA-negative bovine blood samples, and the real-time PCR and LAMP assays
were performed as described above. When a sample was positive or
negative in duplicate analyses, the result was interpreted as a positive
in both assays.
3. Results and Discussion
The results of the conventional and the EZ-Fast extraction methods were
highly consistent. Diagnostic sensitivities and specificities were
92.5% (135/146) and 100% (37/37) for real-time PCR and 93.6%
(132/141) and 100% (42/42) for LAMP (Tables 2 and 3). In contrast to
the conventional automatic DNA extraction platform, which requires 26
min to complete and costs >$3.50 (USD) for DNA extraction
reagents, the EZ-Fast kit required 10 min for crude DNA extraction and
costs <$0.10 (USD) (Fig. 1).
The LOD of EZ-Fast used detection was significantly lower
(>10-fold) than that of the conventional detection method
(Table 4). The LOD of real-time PCR was achieved for the conventional
and the EZ-Fast extraction methods for samples diluted
103-fold. However, at 104-fold
dilution, the conventional method yielded one positive and one negative
in duplicate analyses, although negative in the EZ-Fast extraction.
Compared with the real-time PCR assay, the LAMP assay yielded six
false-negative and one false-positive samples using the conventional
method and three samples using the EZ-Fast kit (Tables 2 and 3), which
is likely explained by the difference between the LODs of the LAMP vs
real-time PCR assay (Table 4).
The average CT value of the positive sample was
2.65-times higher using the EZ-Fast kit, which dilutes blood 1:2 with
SDBS, whereas the conventional method adds 200 µl of whole blood to 50
µl of nuclease-free water. Theoretically, the difference in the
BLV-provirus concentrations between the two methods is 8-fold, which
corresponds to 23 (CT values 3). The
average CT value of the positive sample was 2.65, as
expected. Similarly, in the LAMP assay, the EZ-Fast kit yielded eight
false negatives (Table 3). Although real-time PCR used the EZ-Fast kit
reproducibly amplified target DNA, individual samples may be affected by
different inhibitory effects of blood-derived substances (Schrade et.
al., 2012; Wiemels et. al., 1999; Wilson, 1997). Therefore, a
qualitative interpretation should be employed, regardless of
CT values, suggesting that the developed system is
unsuitable for quantifying copy number.
Among 183 bovine blood samples, the real-time PCR data for one sample
(No. 167) were discrepant (CT 30.63, automatic
extraction and CT 22.48, EZ-Fast extraction) (Table 5
and Supplemental Table 1). Considering the possibility of mixing
samples, we repeated both DNA extraction methods in duplicate using the
blood samples stored at –80 °C. The results were equivalent to those of
the initial analyses: CT 30.37 (conventional automatic
extraction) and CT 24.59 (EZ-Fast extraction). We
therefore tested 10-fold serial dilutions of automatically extracted and
crude DNAs from the latter and evaluated LODs using the real-time PCR
and LAMP assays performed in duplicate (Table 5). In both assays, crude
DNA obtained using the EZ-Fast kit achieved
104–105 greater sensitivity
compared with the conventional automatic extraction method (Table 5).
Although we are unable to explain the reason for this discrepancy, we
determined that more target DNA was contained in the EZ-Fast-extracted
crude supernatant than in DNA automatically extracted from whole blood.
Compared with SDS, SDBS performs equally well for extracting DNA from
cells with reduced inhibitory effects on real-time PCR reactions when
the SDS in the samples is neutralized with 5% Tween 20 (Sekikawa,
2017). Here we were surprised to find that simple heating at 95 °C for 5
min followed by 5-min centrifugation using a portable tabletop
centrifuge operated at full speed (1,260–2,840 x g ) was
sufficient to obtain crude DNA suitable for real-time PCR and LAMP
amplifications, despite significant contamination with blood components.
Further, tedious neutralization using a Tween 20 solution was not
required for reproducible DNA amplification with both amplification
techniques. The developed system may therefore be easily applied to
on-site diagnosis in an epidemic setting for detecting pathogens that
require detection of DNA in blood, including serum and plasma, such as
African swine fever and protozoan diseases.
4. Conclusions
We developed an on-site diagnostic system suitable for epidemic field
settings that does not require advanced laboratory equipment and
expensive DNA extraction kits. This economical, easy, rapid, highly
sensitive and specific system promises to provide an important new tool
for routine veterinary medicine as well as for preventing and combating
devastating pandemics such as that caused by SARS-CoV-2.
Acknowledgments
The authors thank Mari Yamamoto for technical assistance. This research
was supported by JSPS KAKENHI, Grant Number JP18K05535, JSPS Bilateral
Open Partnership Joint Research Projects and AMED under Grant Number
JP19jk0210007.
Data availability Statement
The data that supports the findings of this study are available in the
supplementary material of this article.
Ethics Statement
The authors confirm that the ethical policies of the journal, as noted
on the journal’s author guidelines page, have been adhered to and the
appropriate informed client consent has been received for the sample
collection of bovine blood from client-owned cattle.
Conflict of Interest Statement
The authors declare that they have no conflict of interest.
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