Analytical Biochemistry 658 (2022) 114931

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Development of label-free electrochemical impedance spectroscopy for the

detection of HLA-B*15:02 and HLA-B*15:21 for the prevention of
carbamazepine-induced Stevens-Johnson syndrome
Sirinart Chomean a, c, Nontaya Nakkam b, Wichittra Tassaneeyakul b, Jirapat Attapong a, c,
Chollanot Kaset a, c, *
a
Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand
b
Department of Pharmacology, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
c
Thammasat University Research Unit in Medical Technology and Precision Medicine Innovation, Thailand

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Carbamazepine (CBZ) is an FDA-approved anticonvulsant that is widely used to treat epilepsy, bi­
HLA-B*15:02 polar disorder, trigeminal neuralgia and chronic pain. Several studies have reported a strong association between
EIS HLA-B*15:02 and carbamazepine-induced Stevens–Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN).
SJS
However, the HLA-B75 serotype (HLA-B*15:02, HLA-B*15:08, HLA-B*15:11 and HLA-B*15:21) has been found
TEN
in patients with carbamazepine-induced SJS/TEN.
PCR-SSP
Methods: This study aimed to develop label-free electrochemical impedance spectroscopy (EIS) for the detection
of HLA-B*15:02 and HLA-B*15:21 after PCR-SSP amplification. A total of 208 DNA samples were tested. The
impedance was measured and compared to standard gel electrophoresis.
Results: The developed label-free EIS identified HLA-B*15:02 and HLA-B*15:21 alleles with 100% sensitivity
(95% CI: 86.773%–100.000%) and 95.05% specificity (95% CI: 90.821%–97.714%), comparable to commercial
DMSc 15:02 detection kits.
Conclusions: We successfully developed a novel PCR-SSP associated with signal impedance changes to detect the
HLA-B*15:02 allele and HLA-B*15:21 without downstream amplicon size analysis that is suitable for screening
individuals before indication of CBZ therapy.

1. Introduction
Carbamazepine (CBZ) is widely used to treat epilepsy, bipolar dis­
order, trigeminal neuralgia and chronic pain. However, carbamazepine
can cause many forms of adverse reactions, from mild skin rash to
serious blistering conditions such as Stevens–Johnson syndrome (SJS)
and toxic epidermal necrolysis (TEN). In addition, the US FDA recom­
mended genetic screening of HLA-B*15:02 in patients before initiation
of carbamazepine therapy [1]. An association between HLA-B*15:02
and carbamazepine-induced SJS/TEN was reported in Thai, Han Chi­
nese, Malays, Indians and Vietnamese individuals [2–8]. However, no
association was observed among Koreans, Japanese and Caucasians
[8–11]. In 2018, Capule F et al. reported that a 40-year-old Filipino man
patient who tested positive for the HLA-B75 serotype and genetic testing
was positive for HLA-B*15:21 and negative for HLA-B*15:02 [12].
Moreover, the HLA-B*15:02 allele was not detected in Japanese patients
with carbamazepine-induced SJS/TEN; instead, the HLA-B*15:11 allele
was found [13]. In India, the HLA-B*15:02 allele was detected in six out
of eight patients with carbamazepine-induced SJS, and the HLA-B*15:08
allele was found in one patient [4]. In the Thai population, the
HLA-B*15:02 allele was detected in 37 of 42 patients with
carbamazepine-induced SJS/TEN, and HLA-B*15:21 and HLA-B*15:11
were detected in some patients [5]. Overall, carbamazepine-induced
SJS/TEN may be serotype-specific since HLA-B*15:02, HLA-B*15:08,
HLA-B*15:11 and HLA-B*15:21 are members of the HLA-B75 serotype
[14]. Genetic screening tests have been developed for the detection of
the HLA-B*15:02 allele, including multiplex PCR [1,15], loop-mediated
isothermal amplification (LAMP) [16], and nested PCR methods [17],
which still require downstream amplicon size analysis, such as agarose
gel electrophoresis. Moreover, highly sensitive amplification of the

* Corresponding author. Department of Medical Technology, Faculty of Allied Health Sciences, Thammasat University, Pathumthani, Thailand.
E-mail address: chollanotk@gmail.com (C. Kaset).

https://doi.org/10.1016/j.ab.2022.114931
Received 8 August 2022; Received in revised form 20 September 2022; Accepted 23 September 2022
Available online 1 October 2022
0003-2697/© 2022 Elsevier Inc. All rights reserved.
S. Chomean et al.
LAMP method and the reamplification steps of nested PCR may cause a
high risk of cross-contamination between DNA samples, which leads to
false positive results [18].
To overcome these limitations, electrochemical impedance spec­
troscopy (EIS), one of the most sensitive DNA biosensors, is a promising
technique for the development of HLA-B*15:02 and HLA-B*15:21 allele
detection. It provides high analytical performance, a fast response, cost
effectiveness and label-free detection. A previous study proposed EIS for
DNA or PCR detection by short-synthesized DNA sequences such as
single-stranded DNA probes, target DNA and noncomplementary DNA
[19–22]. A direct measurement of charge transfer resistance (Rct) from
the reactions could allow qualitative and quantitative DNA analysis. To
achieve label-free detection, Hoechst 33258, 20-(4-hydrox­
yphenyl)-5-(4-methyl-1-piperazinyl)-2,50-bi(1H-benzimidazole), was
used to selectively bind to adenine-thymine regions in the minor groove
of DNA [19,21,23]. As a result, the change in the current signal corre­
sponds to the presence and absence of target DNA. A previous study
successfully developed label-free electrochemical DNA biosensors to
directly detect target DNA. Most publications have focused on bacterial
genes, such as Escherichia coli O157:H7, methicillin-resistant Staphylo­
coccus aureus (MRSA), Pseudomonas syringae pv. lachrymans (Psl) and
Salmonella bacteria [24–26]. Due to complexity of human genes, there
are several reports that have used EIS to detect multidrug resistance
genes and telomerase activity for the treatment and prognosis of cancer
[27]. In the present study, a newly designed Polymerase Chain Reaction-
Sequence-specific amplification (PCR-SSP) coupled with label-free
electrochemical impedance spectroscopy was developed to screen
HLA-B*15:02 and HLA-B*15:21 for the prevention of
carbamazepine-induced SJS/TEN in individuals of Asian ancestry. The
proposed method can improve diagnostic efficacy and reduce analytical
time for distinguishing the presence and absence of the HLA-B*15:02
and HLA-B*15:21 alleles.
2. Experimental
2.1. Samples
This study was approved by the Human Research Ethics Committee
of Thammasat University (Science), Thailand (COA No. 116/2561). The
208 volunteers signed consent forms, and blood was collected. Genomic
DNA was extracted from EDTA blood samples using a genomic DNA
extraction kit (Gentra Puregene Blood Kit, QIAGEN, Germany). Genomic
DNA quality and quantity were determined by using a NanoDrop™ One/
OneC Microvolume UV–Vis Spectrophotometer (Thermo Fisher Scien­
tific, USA). The purity of extracted genomic DNA was evaluated by
calculation of the optical density (OD) ratio at 260/280 and 260/230
nm. The extracted genomic DNA was aliquoted and stored at − 20 ◦ C
until use.
Known HLA-B*15:02 genomic DNA samples were provided by Prof.
Dr. Wichittra Tassaneeyakul, Department of Pharmacology, Khon Kaen
University, Khon Kaen, Thailand. The known DNA samples were HLA-
typed using LIFECODES HLA-B eRES SSO Typing (Immucor, Inc. Nor­
cross, GA, USA).
2.2. Primer design
Based on HLA-B alleles present in the Thai population with a fre­
quency greater than one percent [28], DNA sequences of HLA-B*07:05,
13:01/02, 15:02, 15:08, 15:11/12/13, 15:17, 15:21, 15:25,
18:01/18:02, 27:04, 27:06, 35:05, 38:02, 39:01, 40:01/02, 44:02/03,
46:01, 48:03, 51:01/02, 52:01, 54:01, 55:02, 56:01/04, 57:01 and 58:01
were obtained from the IMGT/HLA database with HLA-B*070201 as the
consensus sequence. The related HLA-B allele in serotype B75 is shown in
bold font. DNA alignment was performed using Clustal W (Bioedit version
7.2.6.1), and then primers were designed from exons 2–3, which show
high polymorphism (Fig. S1). The primer sequences consisted of B75F:
2
Analytical Biochemistry 658 (2022) 114931
5′ -GGAGTATTGGGACCGGAAC-3′ and B75R: 5′ -AGCCATACATCCTCTG
GATGA-3′ for amplifying HLA-B*15:02 and the related B75 serotype,
yielding a 375 bp amplicon. In addition, a pair of primers for human
growth hormone (HGH) (1070F: 5′ -GCCTTCCCAACCATTCCCTTA-3′ and
1070R: 5′ -GTCCATGTCCTTCCTGAAGCA-3′ ) was used as an internal
control, yielding a 1070 bp amplicon [29].
2.3. Optimization of in-house PCR-SSP
To set up an in-house PCR-SSP, known HLA-B*15:02/13:01 genomic
DNA samples were used as the positive allele, whereas HLA-B*40:01/
46:01 was used as the negative allele. The optimal in-house PCR-SSP
was performed in a total volume of 25 μL containing extracted genomic
DNA and PCR mixture (12.5 μl of 2X PCR Master mix Solution (i-Taq,
iNtRON, Korea), 5X Q solution (QIAGEN, Germany), HGH1070F and
HGH1070R primers at 2.5 μM, B75F and B75R primers at 10 μM,
nuclease-free water). The in-house PCR-SSP was performed in a T100
Thermal Cycler (Bio-Rad, USA) under optimal conditions: initial pre­
denaturation at 95 ◦ C for 5 min followed by 34 cycles of 95 ◦ C for 1 min,
56 ◦ C for 1 min, 72 ◦ C for 1 min and final extension at 72 ◦ C for 5 min. A
PCR amplicon was separated on a 1.5% agarose gel at 80 V for 50 min
with a 100 bp ladder (Biotechrabbit GmbH, Germany). To test the effi­
cacy of in-house PCR-SSP, 30 known DNA samples were used (Table S1),
and then PCR amplicons were analyzed by gel electrophoresis and
subjected to EIS measurement.
2.4. Optimization of ElS conditions
EIS measurements were performed by potentiostat and galvanostat
with an electrochemical impedance spectroscopy analyzer (Palm­
Sens4TM, Netherlands) using a three-electrode system. The screen
printed carbon electrode (1.0 in width and 3.8 in length mm2 surface
area) served as the working electrode, and a silver chloride (Ag/AgCl)
electrode was used as the counter and reference electrode (Zimmer and
Peacock Limited, Norway).
The optimal concentration of Hoechst 33258 was evaluated to
identify the positive (15:02/13:01) from negative (40:01/46:01) HLA-B
alleles. The final concentration of Hoechst 33258 varied at 0.01, 0.1 and
1.0 mM with 0.01 M phosphate buffer saline (PBS, pH 7.4). Fifty-four
microliters of Hoechst 33258 was mixed with 6 μL of an in-house
PCR-SSP amplicon and dropped on the electrode surface. The imped­
ance parameter was registered at frequencies ranging from 0.1 Hz to
0.01 MHz (47 frequencies) at a constant potential of 300 mV and
alternate potential of 10 mV.
The limit of detection (LOD) was determined based on the lowest
quantity of extracted genomic DNA concentration that could distinguish
the positive (15:02/13:01) from negative (40:01/46:01) alleles. A serial
2-fold dilution generated genomic DNA of 100, 50, 25, 12.5, 6.25, 3.13,
1.56, and 0.78 ng was amplified by in-house PCR-SSP. The PCR-SSP
amplicons were analyzed by agarose gel electrophoresis and EIS mea­
surement. The experiments were carried out by using known positive
(15:02/13:01) and negative (40:01/46:01) alleles in triplicate. Addi­
tionally, distilled water was used as a non-template control (NTC) to
monitor contamination and primer-dimer formation that could produce
false positive results.
2.5. Validation of in-house PCR-SSP
The 208 genomic DNA samples were tested using in-house PCR-SSP
comparable to the commercial DMSc-PGx.1502 kit (Department of
Medical Sciences, Ministry of Public Health, Tivanond Road Nonthaburi
11000, Thailand). Only discrepant results between the two techniques
were typed using the LIFECODES HLA-B eRES SSO typing kit (Lot:
3008560, Immucor, Stamford, CT, USA). The probe-hit pattern was
compared with the common and well-documented (CWD) HLA alleles
Probe Hit Tables (IMGT/HLA Sequence Database Release 3.11.0) by
S. Chomean et al.
using LIFECODES MATCH IT DNA Software version 1.2 based on the
database MatchIT3.39, Allele Database version 3.39 (Immucor). The
performance of the in-house PCR-SSP included sensitivity, specificity,
positive likelihood ratio (+LR), negative likelihood ratio (-LR), positive
predictive value (PPV), negative predictive value (NPV) and allele fre­
quency, which were analyzed by applying standard formulas.
2.6. Clinical application of EIS measurement for HLA-B*15:02 allele
detection
To determine the optimal cutoff points of EIS parameters for the
identification of the HLA-B*15:02 allele, 30 known clinical samples
were evaluated. Genomic DNA was extracted from the blood and
amplified by an in-house PCR-SSP method as previously described. Six
microliters of PCR amplicon was mixed with 54 μL of 0.1 mM H33258 in
0.1 M PBS buffer (pH 7.4). EIS was performed at a fixed frequency of
0.15 Hz with a constant potential of 300 mV and alternate potential of
10 mV. Subsequently, the diagnostic efficacy of EIS measurement for
HLA-B*15:02 allele detection was assessed by using 208 clinical blood
samples. DNA was extracted, and the HLA-B*15:02 allele was amplified
as described above. EIS measurements for identifying the HLA-B*15:02
allele were performed, and impedance shifts were measured. The results
were compared with those of 1.5% agarose gel electrophoresis. Finally,
analysis of the area under the receiver operating characteristic (ROC)
curve (AUC) was performed between the in-house PCR-SSP and DMSc-
PGx.1502.
2.7. Statistical analysis
The Mann-Whitney U test was used to compare EIS values between
positive and negative in-house PCR-SSP. The sensitivity, specificity,
positive predictive value (PPV) and negative predictive value (NPV)
were analyzed using MedCalc v19.2.6 (MedCalc Software bv, Ostend,
Belgium).
3. Results
3.1. Optimization of in-house PCR-SSP
Fig. S2A reveals that only a faint specific PCR amplicon (375 bp) was
amplified. Because the HLA-B*15:02 gene contains 58% GC, it might
affect multiplex PCR. In this study, an internal control and specific PCR
amplicon of the expected size were achieved with an annealing tem­
perature of 56 ◦ C after adding Q solution (QIAGEN, Germany). With
B75F and B75R, a specific band was visualized as a 375 bp amplicon, as
Fig. 1. PCR Amplified products of DNA samples with known HLA specificity. No Template DNA (NTC, distilled water), Negative Control (NC, HLA-B*40:01/46:01)
and Positive Control (PC, HLA-B*15:02/13:01).
3
Analytical Biochemistry 658 (2022) 114931
predicted, while a 1070 bp band was amplified with HGH1070F and
HGH1070R as an internal control gene (Fig. S2B). Thirty known DNA
samples were subjected to optimal PCR conditions, and the results
revealed that only HLA-B*15:02 and HLA-B*15:21 (allele-related posi­
tive HLA-B75 serotype) were amplified (Fig. 1 and Table S1).
3.2. Optimization of ElS conditions
In this work, Hoechst 33258 was used to bind PCR amplicons on
screen-printed carbon electrodes and then measured by electrochemical
impedance spectroscopy (PalmSens4TM, Netherlands). Thus, EIS mea­
surement for HLA-B*15:02 allele detection was verified for its diagnostic
ability using a known genomic DNA sample with positive (15:02/13:01)
and negative (40:01/46:01) alleles. Various Hoechst 33258 concentra­
tions ranging from 0.01 to 1.0 mM were investigated. As shown in Fig. 2,
the impedance represented in Nyquist plots (Fig. 2B), charge transfer
resistance (Fig. 3B), and imaginary impedance (Fig. 3E) could identify
the positive HLA-B*15:02 allele (internal control PCR amplicon at 1070
bp and specific PCR amplicon at 375 bp) from negative (internal control
gene at 1070 bp) and no template control (NTC, distilled water) at 0.1
mM Hoechst 33258. Analysis of Nyquist plots suggested that the imag­
inary impedance (Z”) at a frequency of 0.15 Hz can be applied to di­
agnose the HLA-B*15:02 allele. There were statistically significant
results between the presence and absence of the HLA-B*15:02 allele (p
< 0.001).
To study the limit of detection (LOD), genomic DNA was diluted to
concentrations ranging from 0.78 to 100 ng and amplified by in-house
PCR-SSP. Next, 6 μL of each concentration was mixed with 0.1 mM
Hoechst 33258, and electrical resistances were measured. At a frequency
of 0.15 Hz, the imaginary impedance (Z”) of the HLA-B*15:02 allele was
significantly higher than that of the HLA-B*15:02 allele and NTC
(Fig. 4A). The lowest DNA concentration that could diagnose the HLA-
B*15:02 allele was 6.25 ng. However, analysis by agarose gel electro­
phoresis required at least 12.5 ng (Lane 4, Fig. 4B) of DNA template to be
clearly identified.
3.3. Validation of in-house PCR-SSP
To validate the in-house PCR-SSP assay for the detection of the HLA-
B*15:02 allele and HLA-B*15:21, 208 genomic DNA samples were tested
using our PCR-SSP comparable to the commercial DMSc-PGx.1502 kit
(Department of Medical Sciences, Ministry of Public Health, Tivanond
Road Nonthaburi 11000, Thailand). The performance of the in-house
PCR-SSP revealed 100% sensitivity, 95.05% specificity, +LR of 20.22
(95% CI: 10.70 to 38.24), 78.11% PPV (95% CI: 65.37%–87.09%),
S. Chomean et al.
Fig. 2. EIS measurement (A–C) Nyquist plots and circuits at frequencies
ranging from 0.01 Hz to 10 kHz for 0.01 (A), 0.1 (B) and 1.0 mM (C) Hoechst
33258. Diagnostic ability was evaluated based on the ability to discriminate the
positive HLA-B*15:02 allele from the negative HLA-B*15:02 allele and the
nontemplate control (NTC, distilled water).
100% NPV, and 95.80% accuracy (95% CI: 92.10%–98.09%). However,
8 samples showed discrepant results between the two techniques and
were typed using the LIFECODES HLA-B ERES SSO typing kit (Immucor,
Stamford, CT, USA) (Table 1).
All negative screening results were consistent with the DMSc-
PGx.1502 results. In the in-house PCR-SSP, the results identified 35
positives for HLA-B*15:02 screening out of 208 samples. Of those 35
positive results, three samples with HLA-B*15:13 were false positive
alleles.
Thus, the sensitivity and specificity of the in-house PCR-SSP were
100% and 95.05%, respectively.
4
Analytical Biochemistry 658 (2022) 114931
3.4. Clinical application of EIS measurement for HLA-B*15:02 allele
detection
To develop EIS measurements for HLA-B*15:02 allele detection in
clinical samples, PCR-SSP amplicons from 30 known clinical blood
samples were assessed. The imaginary impedances (Z′′ ) were analyzed to
discriminate the samples with a positive HLA-B*15:02 allele from those
with a negative HLA-B*15:02 allele (Table S1). Fig. S3 shows that the
imaginary impedances (Z”) at 162 KΩ could differentially diagnose the
HLA-B*15:02 allele with 100% sensitivity (95% CI: 86.78%–100%) and
95% specificity (95% CI: 90.82%–97.71%) when compared to the
commercial DMSc-PGx.1502 kit. However, the developed EIS for HLA-
B*15:02 allele detection has 100% sensitivity and a specific method
comparable to standard gel electrophoresis.
For clinical use, the diagnostic potency of EIS measurement for HLA-
B*15:02 allele detection was evaluated in 208 blood samples. All PCR
amplicons from in-house PCR-SSP were measured using Palmsens 4
following optimal conditions as mentioned above. Fig. 5A reveals that a
label-free EIS measurement of the imaginary impedance (Z”) value at
162 KΩ can be used to detect the presence of an internal control gene
and a specific PCR amplicon (presence of the HLA-B*15:02 allele),
which is separate from the internal control PCR amplicon alone (absence
of the HLA-B*15:02 allele) and the NTC. Analysis of the area under the
ROC curve (AUC) was 1.000 (95% CI: 0.982 to 1.000) when compared to
in-house PCR-SSP (Fig. 5B). Overall, the sensitivity and specificity of the
platform were 100% and 95.05%, respectively, when compared to
DMSc-PGx.1502.
4. Discussion
Genetic screening tests have been developed for the detection of the
HLA-B*15:02 allele, including multiplex PCR [2,15], loop-mediated
isothermal amplification (LAMP) [16], and nested PCR methods [17],
which still require downstream amplicon size analysis, such as agarose
gel electrophoresis. Moreover, highly sensitive amplification of the
LAMP method and the reamplification steps of nested PCR may cause a
high risk of cross-contamination between DNA samples, which leads to
false positive results [18].
To overcome these limitations, electrochemical impedance spec­
troscopy (EIS), one of the most sensitive DNA biosensors, is a promising
technique for the development of HLA-B*15:02 and HLA-B*15:21 allele
detection. It provides high analytical performance, a fast response, cost
effectiveness and label-free detection. A previous study proposed EIS for
DNA or PCR detection by short-synthesized DNA sequences such as
single-stranded DNA probes, target DNA and noncomplementary DNA
[19–22]. A direct measurement of charge transfer resistance (Rct) from
the reactions could allow qualitative and quantitative DNA analysis. To
achieve label-free detection, Hoechst 33258, 20-(4-hydrox­
yphenyl)-5-(4-methyl-1-piperazinyl)-2,50-bi(1H-benzimidazole), was
used to selectively bind to adenine-thymine regions in the minor groove
of DNA [19,21,23]. As a result, the change in the current signal corre­
sponds to the presence and absence of target DNA. A previous study
successfully developed label-free electrochemical DNA biosensors to
directly detect target DNA. Most publications have focused on bacterial
genes, such as Escherichia coli O157:H7, methicillin-resistant Staphy­
lococcus aureus (MRSA), Pseudomonas syringae pv. lachrymans (Psl)
and Salmonella bacteria [24–26]. Due to complexity of human genes,
there are several reports that have used EIS to detect multidrug resis­
tance genes and telomerase activity for the treatment and prognosis of
cancer [27]. In the present study, a newly designed polymerase chain
reaction-sequence-specific amplification (PCR-SSP) coupled with
label-free electrochemical impedance spectroscopy was developed to
screen HLA-B*15:02 and HLA-B*15:21 for the prevention of
carbamazepine-induced SJS/TEN. The proposed method can improve
diagnostic efficacy and reduce analytical time for distinguishing the
presence and absence of the HLA-B*15:02 and HLA-B*15:21 alleles.
S. Chomean et al. Analytical Biochemistry 658 (2022) 114931

Fig. 3. EIS measurement (A–C), charge transfer resistance (D–F) and imaginary impedance (Z′′ ) at a frequency of 0.15 Hz for 0.01, 0.1- and 1.0-mM Hoechst 33258.
Diagnostic ability to discriminate the positive HLA-B*15:02 allele from the negative HLA-B*15:02 allele and the nontemplate control (NTC, distilled water) was
evaluated. (* and ** indicate a significant difference compared to NTC and negative HLA-B*15:02 allele, respectively.)

In this study, Fig. S2A reveals that only a faint specific PCR amplicon
(375 bp) was amplified. Because the HLA-B*1502 gene contains 58%
GC, it might affect multiplex PCR. To improve the specificity, sensitivity
or yield of PCR amplicons, the addition of enhancing agents is required,
especially GC-rich DNA templates. Ghasemi [30] found that a combi­
nation of three different PCR enhancing agents (betaine, DMSO, and
7-deaza-dGTP) improved the amplification of DNA sequences contain­
ing 67–79% GC [30]. Templates with moderate GC (56%) were suc­
cessfully amplified after adding Q-Solution into commercial PCR master
mix [31]. Consistent with current study, an internal control and specific
PCR amplicon of the expected size were achieved after adding Q solution
(QIAGEN, Germany) (Fig. S2B).
To validate the in-house PCR-SSP assay for the detection of the HLA-
B*15:02 allele and HLA-B*15:21, 208 genomic DNA samples were tested
using our PCR-SSP comparable to the commercial DMSc-PGx.1502 kit.
For in-house PCR-SSP, the results revealed 35 positives for HLA-B*15:02
screening out of 208 samples. Of those 35 positive results, three samples
with HLA-B*15:13 were false positive alleles. The frequency of HLA-
B*15:13 is 1.8% in Thai populations and varies rarely in Asian
American populations [5]. Other alleles (less than 1%) found in the Thai
population (HLA-B*27:03, 15:32, 35:02, 53:01) were amplified as false
positive results. In 2018, Capule [12] reported that a 40-year-old Fili­
pino man patient who tested positive for the HLA-B75 serotype and
genetic testing was positive for HLA-B*15:21 and negative for
HLA-B*15:02 [12]. Similarly, a significant association was observed
between HLA-B*15:21 and CBZ-SJS [5,18]. Moreover, a strong associ­
ation was found between HLA-B75 serotypes including HLA-B*15:21
and HLA-B*15:11 and CBZ-induced SJS/TEN [32]. Based on our
in-house PCR-SSP, one sample with HLA-B*15:21 (allele frequencies =
0.15% in Thai) was detected, whereas DMSc-PGx.1502 could not detect
this allele.
Regarding possible false positive results, the set of primers used in
our in-house PCR-SSP was not completely matched to the HLA-B allele
with a frequency of more than 1% in the Thai population. The rare allele
HLA-B*15 has been reported as a false positive [16–18]. Heterozygosity
of HLA-B*18 with HLA-B*13:01, 15:13, 15:25 and 15:36 can be detected
as a false positive after the application of a TaqMan probe followed by
SYBR [33]. However, the false positive rate of the HLA detection kit

5
S. Chomean et al. Analytical Biochemistry 658 (2022) 114931

Fig. 4. Limit of detection of EIS measurement (A) compared to that of agarose gel electrophoresis (B) for identifying the HLA-B*15:02 allele. Various concentrations
of genomic DNA were amplified by PCR-SSP and analyzed by both methods; lanes 1–8 represent genomic DNA concentrations of 100, 50, 25, 12.5, 6.25, 3.13, 1.56,
and 0.78 ng, respectively. (*indicates a significant difference compared to negative HLA-B*15:02 allele and NTC.)

HLA-B*15:02 detection at 100 kHz was chosen for differentiation of

Table 1
impedance changes among the positive complementary targets, the
Test comparison between in-house PCR-SSP and commercial DMSc-PGx.1502
positive oligonucleotide and the noncomplementary negative samples
Kit.
[34]. Lim and colleagues detected PCR amplicons using label-free EIS
Sample Name In-House PCR-SSP DMSc-PGx.1502 HLA-B ERES SSO typing
with impedance at 20.02 kHz for imaginary parts [35]. This suggests
004 Positive Negative 27:03/44:03 that using different platform setups and electrode properties may affect
014 Positive Negative 15:13/44:03 the input voltage.
100 Positive Negative 13:01/15:13
For clinical use, the diagnostic potency of EIS measurement for HLA-
NM012 Positive Negative 15:32/35:03
NM036 Positive Negative 35:02/46:01 B*15:02 and HLA-B*15:21 allele detection was evaluated in 208 blood
NM045 Positive Negative 15:21/40:06 samples. All PCR amplicons from in-house PCR-SSP were measured
NM055 Positive Negative 15:01/15:13 using Palmsens 4. Results reveals that a label-free EIS measurement of
NM062 Positive Negative 53:01/58:01
the imaginary impedance (Z”) value can be used to detect HLA-B*15:02
*Only discrepant results between the two techniques were represented. or HLA-B*15:21 allele, which is separate from absence of the HLA-
B*15:02 or HLA-B*15:21 allele and the NTC. This result was sup­
depends on the HLA-B allele frequency in each ethnic group [5]. To this ported by the study of Genetic variants associated with severe cutaneous
point, the use of HLA detection should be considered before imple­ adverse reactions induced by carbamazepine that the alleles in HLA-B75
mentation in a particular setting to ensure that false positives do not serotype (HLA-B*15:02, HLA-B*15:11 and HLA- B*15:21) screening test
occur unexpectedly. For example, HLA-B*15:13 gives false positives in improves the sensitivity for identifying the individuals at the risk of
the PG1502 kit, and HLA-B*15:13 is very rare in East Asia but can developing CBZ-induced SJS/TEN [32].
exceed 0.12 in Malaysia, Indonesia and Singapore [18].
In this work, Hoechst 33258 was used to bind PCR amplicons on 5. Conclusions
screen-printed carbon electrodes and then measured by electrochemical
impedance spectroscopy. In a previous study, a strong interaction be­ According to the positive association between HLA-B*1502, HLA-
tween Hoechst 33258 and dsDNA was proven by fluorescence spec­ B*15:21 and CBZ-SJS, a screening method is required to prevent CBZ-
troscopy and EIS or differential pulse voltammetry (DPV) measurements induced severe hypersensitivity. Routine laboratory PCR testing for
[19]. Conversely, an interdigitated electrode (IDE) biosensor platform HLA typing generally takes 4–5 h to generate results for clinicians but
utilizing loop-mediated isothermal amplification (LAMP-IDE) for the developed method can confirm the HLA-B*1502 and HLA-B*15:21

6
S. Chomean et al.
Fig. 5. Analytical performance of label-free EIS measurement analyzed in 208
clinical blood samples. The imaginary impedance (Z′′ ) value at 162 KΩ (A) and
receiver operating characteristic (ROC) curve analysis of EIS (B).
in 2 h. To reduce the turnaround time, electrochemical impedance
spectroscopy (EIS) was used to discriminate between two PCR amplicons
(positive) and one PCR amplicon (negative) (1 min/samples) instead of
using agarose gel electrophoresis, which is time-consuming (50 min). A
novel PCR-SSP associated with signal impedance changes was used to
detect the HLA-B*15:02 allele and HLA-B*15:21 without downstream
amplicon size analysis, which is suitable for screening individuals for the
prevention of CBZ-induced SJS/TEN.
Author statement
Chollanot Kaset: Conceptualization, Methodology, Validation,
Writing – review & editing, Investigation. Sirinart Chomean: Method­
ology, Validation, Writing – review & editing, Investigation. Wichittra
Tassaneeyakul: Conceptualization, Supervision. Nontaya Nakkam:
Methodology, Investigation. Jirapat Attapong: Investigation, Validation.
All authors have given approval for the final version of the manuscript.
Funder
Thammasat University Research Unit in Medical Technology and
Precision Medicine Innovation
Data availability
Data will be made available on request.
7
Analytical Biochemistry 658 (2022) 114931
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ab.2022.114931.
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