Original Article

Yonago Acta medica 2016;59:262–269

Development of Reverse Transcription Loop-Mediated Isothermal Amplificationfor Simple and Rapid Detection of Promyelocytic Leukemia–Retinoic Acid Receptor α mRNA
Yuki Hashimoto,* Yuki Hatayama,* Nao Kojima,* Shota Morishita,* Satoko Matsumoto,* Yuzuru Hosoda,†‡Ayako Hara* and Toru Motokura*†‡*Division of Clinical Laboratory, Tottori University Hospital, Yonago 683-8504, Japan, †Department of Hematology, Tottori University
Hospital, Yonago 683-8504, Japan and ‡Division of Clinical Laboratory Medicine, Department of Pathophysiological and Therapeutic
Science, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan

ABSTRACT

now been developed as commercial kits, some of whichhave been adopted as officially recommended methods
for routine pathogen examination and surveillance inJapan.5 However, there have been few applications of
LAMP to the field of hematological malignancy, although one notable exception is the detection of Wilms’
tumor-1 gene mRNA.6
Acute promyelocytic leukemia (APL) is a distinct
subtype of acute myeloid leukemia (AML) accounting
for approximately 10% of cases. APL is cytogenetically characterized by balanced reciprocal translocation
between chromosomes 15 and 17, which results in thefusion of the promyelocytic leukemia (PML) and retinoic acid receptor α (RARα) genes.7 The PML-RARα chimeric gene is detected in nearly all APL patients and isused as a marker for clinical diagnosis. In addition, themRNA is detected by reverse transcription polymerase
chain reaction (RT-PCR) and/or real time RT-PCR for
minimal residual disease monitoring of APL.8–10 Threedifferent types of PML-RARα mRNA exist owing todifferent breakpoints in the PML gene, although these
do not result in different treatment outcomes: the long-,
variable-, and short-form types are derived from breakpoints at bcr1 (intron 5), bcr2 (exon 6), and bcr3 (intron3), respectively, and occur at relative frequencies of 55%,
5%, and 40%, respectively.11
APL is the most curable form of AML because ofan established molecular targeted therapy combining alltrans retinoic acid (ATRA) and arsenic trioxide.12 Before
starting the treatment, it is necessary to verify that thepatient has the PML-RARα fusion gene.13 However, early death—mostly due to severe hemorrhage—remains

Background

Acute promyelocytic leukemia (APL) is
a disease characterized by expression of promyelocytic
leukemia–retinoic acid receptor α (PML-RARα) chimeric mRNA. Although APL is curable, early death due
to hemorrhage is a major problem. Here, we report thedevelopment of a simple and rapid diagnostic method for
APL based on reverse transcription loop-mediated isothermal amplification (RT-LAMP).
Methods An RT-LAMP primer set was designed todetect three types of PML-RARα mRNA in a single
reaction. Serial dilutions of plasmid DNA containing
bcr1, bcr2, or bcr3 PML-RARα sequences and RNA
extracted from bone marrow aspirates of 6 patients with
APL were used to compare the results of RT-LAMP andnested PCR assays.
Results Plasmid DNA was amplified by RT-LAMP,
for which the reaction time was > 4 h shorter and thelower detection limit was higher than for nested RTPCR. Six of 7 samples tested positive by both methods.
Conclusion We developed an RT-LAMP assay forsimple and rapid PML-RARα mRNA detection that may
be clinically useful for point-of-care testing and APL
diagnosis.
Key words acute promyelocytic leukemia; polymerase

chain reaction; promyelocytic leukemia–retinoic acidreceptor α; reverse transcription loop-mediated isothermal amplification; t(15;17) chromosome translocation

Loop-mediated isothermal amplification (LAMP) is anestablished nucleic acid amplification method that requires a set of 4 specially designed, highly specific innerand outer primers.1 Additional loop primers can further
accelerate the reaction.2 The LAMP product is detectedby turbidity arising from magnesium pyrophosphate
formed during the reaction.3 This method is very rapidand directly amplifies the target region from an RNA
sample, and has been used to detect various pathogens,including bacteria and viruses.4 In addition, LAMP has

Corresponding author: Yuki Hashimoto
hatsu-1218@umin.ac.jp
Received 2016 August 19
Accepted 2016 October 6Abbreviations: AML, acute myeloid leukemia; APL, Acute promyelocytic leukemia; ATRA, all-trans retinoic acid; LAMP, loop-mediated isothermal amplification; PML, promyelocytic leukemia;
RARα, retinoic acid receptor α; RIN, RNA integrity number; RTLAMP, reverse transcription loop-mediated isothermal amplification; RT-PCR, reverse transcription polymerase chain reaction262

RT-LAMP for rapid detection of PML-RARα mRNA

the main cause of APL treatment failure according torecent population-based studies carried out in developed
countries.14 Some reports recommend early and timely
initiation of ATRA and provision of aggressive supportive care at the first suspicion of the disease, without
awaiting molecular confirmation, in order to reduce
hemorrhagic deaths in APL.14 An improved PML-RARαmRNA detection test is also needed.
We report here the development of a simple reverse
transcription loop-mediated isothermal amplification(RT-LAMP) method for rapid PML-RARα mRNA detection. The advantages of this method with respect to
RT-PCR are also discussed.

assays. The study protocol was approved by the Tottori
University Hospital ethics committee (number 2326).
Primer design

Primer sets for detection of PML-RARα mRNA by RTLAMP (Table 1) were designed using PrimerExplorerver. 4 software (Eiken Chemical, Tokyo, Japan). The
primer sets were modified to detect the three types of
PML-RARα mRNA in a single tube—i.e., bcr1 and bcr2
were amplified with the same primer sets and an additional set consisting of FIP, F3, and LF were included in
one reaction to amplify bcr3 (Fig 1).
RT-LAMPThe RT-LAMP assay was carried out using the Loopamp RNA Amplification kit (Eiken Chemical, Tokyo,
Japan) under the following conditions: 1 μL sample (RNAor DNA) was mixed with 20 pmol each of FIP and BIP
primers, 10 pmol each of LF and LB primers, 2.5 pmol
each of F3 and B3 primers, 1 μL Enzyme Mix, and 12.5
μL of 2 × Reaction Mix, with distilled water added toobtain a final volume of 25 μL. For real-time turbidity
monitoring, the reaction mixture was incubated at 68 °C
for 60 min in an RT-160C Loopamp Realtime Turbidimeter (Eiken Chemical). The specificity of LAMP was
confirmed by restriction enzyme digestion using DdeI
(Takara Bio, Otsu, Japan) under the following conditions: 5 μL LAMP product was mixed with 2 μL of 10
× K buffer (Takara Bio), 1 μL DdeI, with distilled water
added to obtain a final volume of 20 μL, followed byincubation at 37 °C for 120 min. DdeI was inactivated
at 70 °C for 15 min and the digestion products (10-μL
aliquots) were mixed with loading buffer (Nippon Gene,
Toyama, Japan) and separated by 1.5% agarose gelelectrophoresis (100 V, 35 min) with 0.005% ethidium
bromide using a Mupid mini gel electrophoresis system(Advance, Tokyo, Japan), with 5 μL Gene Ladder 100(Nippon Gene) used as a molecular size marker.

MATERIALS AND METHODS
SamplesThree different plasmids—containing bcr1, bcr2, andbcr3 PML-RARα mRNA sequences—were purchasedfrom Eurofins Genomics (Louisville, KY) for use as
positive controls. Vector plasmid was pTAKN-2 andchimeric sequences were made of sequences derived
from the Genbank sequence database (Accession numbers are M73778, NM_000964, and AB067754). IVS0035 RNA (Invivoscribe Technologies, San Diego, CA)
was used as a negative control. Clinical samples used inthis study were archived specimens from APL patientsdiagnosed by detection of APL cells and PML-RARαmRNA by nested RT-PCR at our hospital. RNA wasextracted from patient bone marrow aspirates using the
RNeasy Plus Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions; these were used
for diagnosis, and then frozen at −80 °C until the study.
RNA concentrations were measured and 260/280 nm
absorbance ratios were determined with a Nanodrop
2000 spectrophotometer (Thermo Fisher Scientific,
Waltham, MA) prior to RT-LAMP and nested RT-PCR
Table 1. Primers for detection of PML-RARα mRNA
by RT-LAMP
Primers

Nested RT-PCR

Sequence (5’ → 3’)

The RT reaction was carried out using1 μL RNA sample
and the ThermoScript RT-PCR system (Thermo Fisher
Scientific) according to the manufacturers’ protocol.
Three types of PML-RARα mRNA were detected using
two forward primer sets designed according to publishedsequences.10 Specifically, bcr1 and bcr2 types were
amplified with the same primer set. The PCR reaction
mixture was prepared using AmpliTaq Gold with 10 ×
PCR Gold Buffer and MgCl2 (Thermo Fisher Scientific)
and the GeneAmp dNTP mix (Thermo Fisher Scientific)
under the following conditions: 1 μL plasmid DNA, 2
μL cDNA, or 1 μL first PCR product was mixed with

bcr1,2 FIP TCCCCTCCTCAGACTCCATCTTGAAGAGGAAGTGCAGCCAGA
bcr1,2 F3 CTCCAATACAACGACAGCC
bcr3 FIP TTGAACTCGTCGAAGCCATCGGCTGGACATGCACGGTT
bcr3 F3
AAGTGCTACGCCTCGGABIPTGAGACCCAGAGCAGCAGTTGTAGATGCGGGGTAGAB3TGACAGACAAAGCAAGGC
bcr1,2 LF GATGACCTTCCTGGGGCA
bcr3 LFACGGCAGCTTGCAGGCTLBTGAAGAGATAGTGCCCAGCCCTPML-RARα, promyelocytic leukemia–retinoic acid receptor α;
RT-LAMP, reverse transcription loop-mediated isothermal amplification.

263

Y. Hashimoto et al.

PML

RARα

DdeI

bcr1,2 FIP

DdeI

DdeI

bcr1,2 F3

bcr 1

3

4

5

6

3

B3
bcr1,2 FIP

BIP

bcr1,2 F3

bcr 2

3

4

5

6

3
B3
BIP

bcr3 FIP
bcr3 F3

bcr 3

3

3
B3
BIP

Fig. 1. Schematic representation of primer design for detection of PML-RARα mRNA by RT-LAMP and cutting sites of DdeI restriction
enzyme assay. Exon numbers of the PML gene (dark) and the RARα gene (light) are shown. PML, promyelocytic leukemia; RARα, retinoic acid receptor α; RT-LAMP, reverse transcription loop-mediated isothermal amplification.

2.5 μL 10 × PCR Gold buffer, 2.25 nmol MgCl2 solution, 500 pmol dNTP mix, 0.2 μL AmpliTaq Gold DNA
polymerase, and 0.25 pmol each forward and reverse
primers, with distilled water added to obtain a final
volume of 25 μL. The forward primers were as follows:
5’-AGTCAGTGCCCGGGGCACAC-3’ for bcr1 andbcr2 and 5’-AGCTGCTGGAGGCTGTGGACG-3’ forbcr3 in the first PCR; and 5’-AGTGTACGCCTTCTCCATCAAAG-3’ for bcr1 and bcr2 and 5’-TGTGCTGCAGCGACTCCGCA-3’ for bcr3 in the second PCR.
The reverse primers were as follows: 5’-AGGGCTGGGCACTATCTCTTC-3’ for bcr1, bcr2, and bcr3 in thefirst PCR; and 5’-CAGAACTGCTGCTCTGGGTCTCAAT-3’ for bcr1, bcr2, and bcr3 in the second PCR.
Reaction conditions on the MultiGene Mini Personal
Thermal Cycler (Labnet International, Edison, NJ) wereas follows: 94 °C for 5 min; 35 cycles at 94 °C for 30 s,
62 °C for 1 min, and 72 °C for 1 min; and 72 °C for 7
min. The nested PCR products (10-μL aliquots) weremixed with loading buffer and separated along with 5
μL of molecular size marker by 1.5% agarose gel electrophoresis (100 V, 35 min) with 0.005% ethidium bromide.

for which the amount was insufficient) was evaluated by
RIN as previously described15 on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
RESULTS
Specificity of amplification by RT-LAMP

We performed the RT-LAMP assay with two concentrations of plasmid DNA followed by DdeI restriction
enzyme analysis. All plasmid DNAs were detected by
RT-LAMP in 60 min (data not shown, Fig. 2A) andyielded the expected products after digestion with DdeI
(Fig. 3), which differed for each breakpoint. Non-specific amplification products obtained after 60 min from
PML-RARα-negative RNA were not digestible by DdeI
(Fig. 3).
Sensitivity of the RT-LAMP assay

The lower detection limit of the RT-LAMP assay wasdetermined using10-fold serial dilutions of bcr1, bcr2,
and bcr3 plasmid DNA; the result was compared to thatobtained by nested RT-PCR. RT-LAMP was able to detect 880, 78, and 800 copies/tube of bcr1, bcr2, and bcr3
plasmid, respectively (Fig. 2A), as compared to 0.88,
7.8, and 80 copies/tube, respectively, by nested RT-PCR(Figs. 4A–C). The specificity of all RT-LAMP products
was confirmed by DdeI restriction enzyme digestion.

RNA integrity number (RIN) analysis

The quality of clinical RNA samples (expect for S-4,
264

RT-LAMP for rapid detection of PML-RARα mRNA

(A)

(B)

0.25

0.25

bcr1

0.15

0.15

-0.05
0.25

Diluted plasmid DNA
log10 (DF)
ー：2
ー：3
ー：4
ー：5
ー：6
ー：7
ー：8
ー：IVS-0035 RNA
ー：DW

bcr2

0.15
0.05
-0.05
0.25

bcr3

0.15

Turbidity

0.05

0.05

Turbidity

bcr1

-0.05
0.25
0.15
0.05
-0.05 0

0.15
0.05
2000

3000

4000

2000

3000

ー：S-1
ー：S-2
ー：S-3
ー：S-4
ー：bcr1 plasmid DNA
ー：S-5
ー：S-6
ー：S-7
ー：bcr3 plasmid DNA
ー：IVS-0035 RNA
ー：DW

-0.05
0.25

1000

1000

Time (s)0.05-0.05 0

bcr3

5000

Time (s)Fig. 2. Detection of PML-RARα in plasmid DNA and clinical RNA samples by RT-LAMP. (A) Ten-fold serial dilutions of bcr1, bcr2, andbcr3 plasmid DNAs were used as template; the starting amounts were 8.8, 7.8, and 8.0 × 108 copies/tube, respectively. DF of the indicatedplasmid DNA is shown as a logarithmic value. (B) Clinical samples from APL patients (S-1 through -7) were analyzed by RT-LAMP.
IVS-0035 RNA and distilled water were used as negative controls. A 100-fold dilution of corresponding plasmid DNAs served as a positive control. The amplification reaction was carried out at 68 °C for indicated times. APL, acute promyelocytic leukemia; DF, dilution
factor; PML-RARα, promyelocytic leukemia–retinoic acid receptor α; RT-LAMP, reverse transcription loop-mediated isothermal amplification.

M
Dde I

500
400
300
200
100

LAMP
bcr1
bcr2

−

+

−

+

Dde*I

bcr3 neg RNA− + − +

DW
−
+

M

500
400
300

200
100

********M*****b1*****b1*****b2*****b2*****b3*****b3*****N******N*****DW***DW****M*265**Dde*I*:**(*E*)****(*+*)***(*E*)***(*+*)***(*E*)***(*+*)***(*E*)**(*+*)***(*E*)****(*+*)

Fig. 3. DdeI restriction enzyme digestionof RT-LAMP products. Products of bcr1,
bcr2, and bcr3 plasmid DNA amplification
were incubated with (+) or without (−)
DdeI at 37 °C for 120 min, followed bydeactivation at 70 °C for 15 min and separation by 1.5% agarose gel electrophoresis(100 V, 35 min). The amounts of bcr1,
bcr2 and bcr3 plasmid DNAs were 8.8,
7.8, and 8.0 × 106 copies/tube, respectively.
The expected sizes of digested RT-LAMP
products are the followings: 468 bp, about
234 bp (stem and loop structure) and 83 bp
for bcr1; 232 bp, about 116 bp (stem andloop structure) and 83 bp for bcr2; and 139
bp and 121 bp (merged in Figure) for bcr3.
DW, distilled water (negative control); M,
100-bp ladder size marker; neg RNA, IVS0035 RNA (negative control); RT-LAMP,
reverse transcription loop-mediated isothermal amplification.

Y. Hashimoto et al.

(A)

(B)

(C)

log10 (DF)
M
0
1
2
3
4
5
6
7
8
9 10 DW))M)))))))1))))))))2))))))))3)))))))4))))))))5)))))))6)))))))7))))))))8))))))))9))))))10))))))11)))))DW

11)tubeM0 10)tube
12
3
45
LAMP bcr1 880)copies/tube

M

0

1

2

3

4

5

6

6

7

7 0.88)copies/tube
8
9 10 11 DW
1000

8

9 10 11 12 13 14 DW

Fig. 4. Nested RT-PCR amplification
using 10-fold serial dilutions of plasmid DNA. Plasmid DNAs containing (A) bcr1, (B) bcr2, and (C) bcr3
sequences (starting amounts: 8.8 ×
108, 7.8 × 108, and 8.0 × 108 copies/
tube, respectively) were diluted by theindicated DF and amplified by nested
RT-PCR. Amplification products
were separated by 1.5% agarose gelelectrophoresis (100 V, 35 min). DF,
dilution factor; DW, distilled water; M,
100-bp ladder size marker; RT-PCR,
reverse transcription polymerase
chain reaction.

Table 2. Comparison between RT-LAMP and nested RT-PCR using clinical samples
Sample number

Breakpoint typeRNA concentration Absorbance ratio(ng/μL)(260/280 mm)

S-1
S-2
S-3
S-4
S-5
S-6
S-7

bcr1
bcr1
bcr1
bcr1
bcr3
bcr3
bcr3

250.8
535.0
361.9
1141.0
389.8
309.2
59.9

2.04
2.04
1.96
2.06
2.06
2.05
2.04

RIN value

RT-LAMP

NestedRT-PCR

9.60
10
NM
NA
9.50
9.90
8.50

+
+
–
+
+
+
+

+
+
–
+
+
+
+

NA, not available; NM, not measured due to pattern deformation; RIN, RNA integrity number; RT-LAMP, reverse transcription loop-mediated isothermal amplification; RT-PCR, reverse transcription polymerase chain reaction; Samples 5 and 6 were derived from the same
patient: S-5, at the initial visit and S-6, at the time of relapse.

266

RT-LAMP for rapid detection of PML-RARα mRNA

(A)

M
Dde I

(B)

S-1

−

+

M
Dde I

S-5

−

+

Fig. 5. Confirmation of bcr1 and bcr3 of clinical samples after RT-LAMP assay. The RT-LAMP products of clinical sample RNAs of
S-1 (A) and S-5 (B) were incubated with (+) or without (−) DdeI at 37 °C for 120 min, followed by deactivation at 70 °C for 15 min andseparation by 1.5% agarose gel electrophoresis (100 V, 35 min). M, 100-bp ladder size marker; RT-LAMP, reverse transcription loop-mediated isothermal amplification.

DISCUSSION
Each type of PML-RARα sequence was specifically
amplified by RT-LAMP; moreover, each PML-RARα
breakpoints as well as non-specific amplification could
be distinguished by digesting the products with DdeI.
The sensitivity of the RT-LAMP assay was inferior tothat of nested RT-PCR by 10 fold for bcr2 and bcr3 andby 1000 fold for bcr1. Some RT-LAMP assays havehigher sensitivity than nested RT-PCR,17–20 while others
have lower sensitivity.21, 22 This depends on the primers
used, which determine the amplification efficiency andhas a significant effect on the sensitivity of gene amplification. In this study, we selected 5 primer sets from
more than 1000 candidates listed by PrimerExplorerver. 4. It is virtually impossible to test all candidates, although a primer set with better amplification efficiency

RT-LAMP of clinical samples

Clinical samples were analyzed by RT-LAMP and nested RT-PCR; these included four bcr1- and three bcr3type PML-RARα RNAs. The plasmid DNAs were usedas positive controls, and IVS-0035 RNA and distilledwater were used as negative controls. Three bcr1- andthree bcr3-type RNAs were detected by RT-LAMP (Fig.
2B); the products were confirmed by DdeI restriction enzyme digestion (Fig. 5), and the results were the same as
those obtained by nested RT-PCR (Table 2). One sample
yielded no product with both assays; this RNA had been
stored for the longest period (about 9 years) and was
likely degraded, as indicated by RIN analysis of RNA
quality.15, 16

267

Y. Hashimoto et al.

may exist.
RT-LAMP and nested RT-PCR yielded similar
results in the analysis of clinical samples. This suggests that RT-LAMP has clinical utility as a diagnostic
test, especially at initial presentation when a sufficientamount of PML-RARα mRNA is available.
The nested RT-PCR assay requires about 6 h fromRNA extraction to target mRNA detection. In contrast,
PML-RARα mRNA was amplified in 30 min by RTLAMP, and is therefore more useful for early diagnosis
and determination of an appropriate intervention. In addition, the amount of sample that is lost with RT-LAMP
is minimal because the extracted RNA is used directlyin the amplification reaction even in one tube. This simplifies the procedure, thereby reducing the labor and costof laboratory testing.
Spinelli et al. recently reported another assay using
RT-LAMP to detect PML-RARα chimeric gene.23 Using fluorescence, the assay appeared to be more rapid
and sensitive than our assay. However, the detection offluorescence requires a dedicated devise and their assay
needs 2 reaction tubes to detect 3 PML-RARα mRNAs. In addition, primer sequences were not described.
Therefore, our assay has advantages of simple procedureand low cost, and could readily be applied in developing
countries. Such simplicity can lead to the developmentof an automated system that can be used for point-ofcare testing and diagnosis of APL.
In conclusion, we developed an RT-LAMP assay
for detection of PML-RARα mRNA in clinical samples
from APL patients. This assay is more simple and rapid
than nested RT-PCR, and can contribute to early diagnosis of APL.

DNA (LAMP): a new diagnostic tool lights the world of diagnosis of animal and human pathogens: a review. Pak JBiol Sci.
2014;17:151-66. PMID: 24783797.
5 Mori Y, Notomi T. Loop-mediated isothermal amplification(LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J Infect Chemother. 2009;15:62-9.
PMID: 19396514.
6 Morishita S, Tani H, Kurata S, Nakamura K, Tsuneda S,
Sekiguchi Y, et al. Real-time reverse transcription loop-mediated isothermal amplification for rapid and simple quantification of WT1 mRNA. Clin Biochem. 2009;42:515-20. PMID:
19297684.
7 Kak izuka A, Miller WH, Umesono K, War rell R P,
Frankel SR, Murty VV, et al. Chromosomal translocationt(15;17) in human acute promyelocytic leukemia fuses RAR
alpha with a novel putative transcription factor, PML. Cell.
1991;66:663-74. PMID: 1652368.
8 Miller WH, Kakizuka A, Frankel SR, War rell RP,
DeBlasio A, Levine K, et al. Reverse transcription polymerase
chain reaction for the rearranged retinoic acid receptor alphaclarifies diagnosis and detects minimal residual disease inacute promyelocytic leukemia. Proc Natl Acad Sci USA.
1992;89:2694-8. PMID: 1372989.
9 van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E,
Rossi V, Saglio G, et al. Standardized RT-PCR analysis of
fusion gene transcripts from chromosome aberrations in acuteleukemia for detection of minimal residual disease. Report ofthe BIOMED-1 Concerted Action: investigation of minimal
residual disease in acute leukemia. Leukemia. 1999;13:190128. PMID: 10602411.
10 Tobal K, Liu Yin JA. RT-PCR method with increased sensitivity shows persistence of PML-RARA fusion transcripts
in patients in long-term remission of APL. Leukemia.
1998;12:1349-54. PMID: 9737682.
11 Gallagher RE, Willman CL, Slack JL, Andersen JW, Li YP,
Viswanatha D, et al. Association of PML-RAR alpha fusionmRNA type with pretreatment hematologic characteristics but
not treatment outcome in acute promyelocytic leukemia: anintergroup molecular study. Blood. 1997;90:1656-63. PMID:
9269786.
12 Lo-Coco F, Avvisati G, Vignetti M, Thiede C, Orlando SM,
Iacobelli S, et al. Retinoic acid and arsenic trioxide for acutepromyelocytic leukemia. New Engl JMed. 2013;369:111-21.
PMID: 23841729.
13 Wang ZY, Chen Z. Acute promyelocytic leukemia: from highly fatal to highly curable. Blood. 2008;111:2505-15. PMID:
18299451.
14 Breccia M, Coco FL. Thrombo-hemorrhagic deaths in acutepromyelocytic leukemia. Thromb Res. 2014;133:S112-S6.
PMID: 24862130.
15 Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M,
Gassmann M, et al. The RIN: an RNA integrity number forassigning integrity values to RNA measurements. BMC Mol
Biol. 2006;7:3. PMID: 16448564.
16 Imbeaud S, Graudens E, Boulanger V, Barlet X, Zaborski P,
Eveno E, et al. Towards standardization of RNA quality assessment using user-independent classifiers of microcapillaryelectrophoresis traces. NucleicAcids Res. 2005;33:e56. PMID:
15800207.
17 Lee S, Kim JH, Choi JY, Jang WC. Loop-mediated isothermal amplification assay to rapidly detect wheat streak mosaic
virus in quarantined plants. Plant Pathol J. 2015;31:438-40.
PMID: 26674930.

Acknowledgments: The authors would like to thank Mr. Yasuharu
Sasaki for technical advice and Editage (www.editage.jp) for English editing.
This study was supported by a grant from Seeds Research Project at Tottori University Hospital.
The authors declare no conflict of interest.

REFERENCES
1 Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe
K, Amino N, et al. Loop-mediated isothermal amplificationof DNA. Nucleic Acids Res. 2000;28:E63. PMID: 10871386.
2 Nagamine K, Hase T, Notomi T. Accelerated reaction byloop-mediated isothermal amplification using loop primers.
Mol Cell Probes. 2002;16:223-9. PMID: 12144774.
3 Mori Y, Nagamine K, Tomita N, Notomi T. Detection ofloop-mediated isothermal amplification reaction by turbidity
derived from magnesium pyrophosphate formation. Biochem
Biophys Res Commun. 2001;289:150-4. PMID: 11708792.
4 Dhama K, Karthik K, Chakraborty S, Tiwari R, Kapoor S,
Kumar A, et al. Loop-mediated isothermal amplification of268

RT-LAMP for rapid detection of PML-RARα mRNA
18 Zhang Q, Liu S, Yang H, Zhu L, Wan X, Li X, et al. Reverse
transcription loop-mediated isothermal amplification for rapid
and quantitative assay of covert mortality nodavirus in shrimp.
J Invertebr Pathol. 2015; Sep 9. doi: 10.1016/j.jip.2015.09.001.
PMID: 26363123.
19 Liu DF, Liu CG, Tian J, Jiang YT, Zhang XZ, Chai HL, et al.
Establishment of reverse transcription loop-mediated isothermal amplification for rapid detection and differentiation of canine distemper virus infected and vaccinated animals. InfectGenet Evol. 2015;32:102-6. PMID: 25769803.
20 A r u n r ut N, Sueb si ng R , Wit hya chu m n a r n k u l B,
Kiatpathomchai W. Demonstration of a very inexpensive,
turbidimetric, real-time, RT-LAMP detection platform using
shrimp Laem-Singh virus (LSNV) as a model. PLoS ONE.

2014;9:e108047. PMID: 25255231.
21 Hanaki K, Ike F, Hatakeyama R, Hirano N. Reverse transcription-loop-mediated isothermal amplification for the detectionof rodent coronaviruses. J Virol Methods. 2013;187:222-7.
PMID: 23123121
22 Kiatpathomchai W, Jareonram W, Jitrapakdee S, Flegel TW.
Rapid and sensitive detection of Taura syndrome virus by reverse transcription loop-mediated isothermal amplification. J
Virol Methods. 2007;146:125-8. PMID: 17643501.
23 Spinelli O, Rambaldi A, Rigo F, Zanghì P, D’Agostini E, Amicarelli G, et al. Simple, rapid and accurate molecular diagnosisof acute promyelocytic leukemia by loop mediated amplification technology. Oncoscience. 2015;2:50-8. PMID: 25815362.

269