Aquaculture International (2021) 29:527–538

https://doi.org/10.1007/s10499-020-00639-5

Ammonium sulfate improves sensitivity and avoids

false negatives of polymerase chain reaction (PCR)
for scale drop disease virus (SDDV) detection

Naruporn Rungrueng 1 & Watcharachai Meemetta 2 & Kornsunee Phiwsaiya 2,3 &
Ha Thanh Dong 1 & Wattana Panphut 1 & Saengchan Senapin 2,3

Received: 25 February 2020 / Accepted: 16 December 2020 / Published online: 13 January 2021
# The Author(s), under exclusive licence to Springer Nature Switzerland AG part of Springer Nature 2021

Abstract
Accuracy is crucial for polymerase chain reaction (PCR) diagnostic laboratories. We
noticed inconsistent PCR results or false negatives that sometimes occurred when
changing commercial brand of Taq DNA polymerase enzyme. Here, we tested 6 primer
sets specific for 3 important aquaculture viruses (scale drop disease virus (SDDV), tilapia
lake virus (TiLV), and white spot syndrome virus (WSSV)) using 7 brands of Taq DNA
polymerase enzymes (RBC Bioscience, Invitrogen, PCRBiosystems, Bio-Helix,
biotechrabbit, GeneAll, and New England BioLabs) with their original reaction buffers.
False negative detection and variation in sensitivity of 10–1000 times were observed.
Interestingly, only enzymes from RBC Bioscience and Invitrogen could amplify SDDV
252 bp products, while 5 other brands failed to do so. The use of reaction buffer from
RBC Bioscience with Taq polymerase enzymes from other brands resulted in improve-
ment of detection sensitivity and avoided false negatives. Comparison of ingredients of
the 7 reaction buffers indicated that bovine serum albumin (BSA) and ammonium sulfate
were present in the RBC Bioscience buffer but absent or not specified in 6 other brands.
Subsequently, we found that adding ammonium sulfate to the original buffers of the 4
selected brands improved detection efficiency for most test reactions, while adding BSA
alone or BSA plus ammonium sulfate showed no or little improvement. When tested with
serial dilutions of DNA extracted from 2 SDDV-infected fish, adding ammonium sulfate

* Wattana Panphut
[email protected]
* Saengchan Senapin
[email protected]

1
Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok, Thailand
2
Fish Health Platform, Center of Excellence for Shrimp Molecular Biology and Biotechnology
(Centex Shrimp), Faculty of Science, Mahidol University, Bangkok, Thailand
3
National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and
Technology Development Agency (NSTDA), Pathumthani, Thailand
528 Aquaculture International (2021) 29:527–538

into the original buffer of biotechrabbit brand could improve detection sensitivity 100
times, equal to the result of RBC Bioscience kit. We thus recommend that PCR reaction
buffer with ammonium sulfate (10 mM) added might improve detection sensitivity and
avoid false negative results in PCR detection assays.

Keywords Ammonium sulfate . False negative . Sensitivity . PCR . SDDV .

Scale drop disease virus

Introduction

Infectious pathogens causing disease outbreaks are one of the major contributors to production loss
in aquaculture (Thitamadee et al. 2016; Jansen et al. 2018; Machimbirike et al. 2019). For example,
scale drop disease virus (SDDV), a newly emerging virus of Asian sea bass (Lates calcarifer)
aquaculture in Southeast Asia, caused 40–50% mortality in infected fish populations (Gibson-
Kueh et al. 2012; de Groof et al. 2015; Senapin et al. 2019; Nurliyana et al. 2020). Similarly, tilapia
lake virus (TiLV) is widely reported in tilapia (Oreochromis spp.) farming countries, which
reportedly caused up to 90% mortality in natural outbreaks (Eyngor et al. 2014; Ferguson et al.
2014; Jansen et al. 2018). For penaeid shrimp, white spot syndrome virus (WSSV) has been the
most devastating pathogen of shrimp aquaculture industry worldwide (Thitamadee et al. 2016).
Polymerase chain reaction (PCR) has become a routine diagnostic method for aquatic
pathogens in aquaculture laboratories. PCR has also been used as an important tool for
screening of pathogen-free animals before stocking to prevent pathogen introduction to
cultured system. Therefore, accurate testing result is crucial to farmers to avoid unexpected
introduction of pathogen(s) to aquaculture system and disease outbreaks (Adams and
Thompson 2011; Thitamadee et al. 2016; Jansen et al. 2018).
We and some other diagnostics laboratories have experienced false negative result or incon-
sistency (i.e., difference in detection sensitivity) when using different brands of Taq DNA
polymerase among laboratories and when employing brands of Taq polymerase that differed
from the originally established PCR protocols. In fact, there are many commercial brands of Taq
DNA polymerase that have been used, and their accessibility might be diverse in different
countries or regions. Thus, similar problem might have happened in numerous molecular
diagnostic laboratories elsewhere. This study demonstrates difference in PCR detection results
when using different brands of Taq DNA polymerase. Comparison was performed using 7 brands
of Taq enzyme and 6 primer sets targeting 3 aquatic viruses: scale drop disease virus (SDDV),
tilapia lake virus (TiLV), and white spot syndrome virus (WSSV). The findings suggested that
adding ammonium sulfate in reaction buffer can reduce false negatives for SDDV PCR diagnosis.

Materials and methods

Plasmid construction

Recombinant pGEM T-easy based plasmids containing viral DNA fragments were used as
PCR templates. The DNA fragments included ATPase gene (738, 252, and 135 bp) of scale
drop disease virus (SDDV), a 460 bp-MCP (major capsid protein) gene fragment of SDDV, a
250-bp product of tilapia lake virus (TiLV) genome segment 3, and a 141-bp VP28 gene
Aquaculture International (2021) 29:527–538 529

fragment of white spot syndrome virus (WSSV). Primers used for PCR amplification are listed
in Table 1. Amplification conditions of SDDV DNA fragments followed our published
protocol (Charoenwai et al. 2019). In brief, a reaction volume of 25 μl contained 0.5 μl of
each 10 μM primer, 0.5 μl of 10 mM dNTPs (Thermo Fisher Scientific), 0.2 μl of Taq DNA
polymerase (RBC Bioscience, Taiwan, 5 units/μl), 200 ng of DNA template derived from
SDDV-infected Asian sea bass tissues, and 1X supplied reaction buffer. The PCR conditions
were denaturation at 94 °C for 5 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s,
and 72 °C for 30 s with a final extension at 72 °C for 5 min. For WSSV VP28 141 bp
amplification, the PCR protocols were modified from Mendoza-Cano and Sánchez-Paz (2013).
Reaction mixture was similar to that of SDDV PCR reactions except that WSSV primers
(Table 1) and DNA extracted from WSSV-infected whiteleg shrimp were used (Sangsuriya
et al. 2014). Themocycling conditions were also followed that of the SDDV DNA amplifica-
tion. Amplification of a 250 bp partial segment 3 of TiLV genome was carried out by RT-PCR
protocol modified from Kembou Tsofack et al. (2016) as described in Dong et al. (2017). A
25 μl reaction volume consisted of 1 μl of each 10 μM primer, 0.5 μl of SuperScript One-Step
RT/Platinum Taq mix (Invitrogen, USA), 200 ng of RNA template extracted from TiLV-
infected tilapia specimen, and 1X supplied reaction buffer. RT-PCR reaction was performed at
50 °C for 30 min, 94 °C for 2 min, and 25 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for
30 s. After agarose gel electrophoresis, expected band products were gel-purified using a

Table 1 Primers used in this study

Virus Gene Product Primer name Primer sequence Reference

(bp) (5′ to 3′)

Scale drop disease ATPase 135 qSDDV-AF AATGACCGAAATAC Sriisan et al. (2020)
virus (SDDV) GACCGAGAAC
qSDDV-AR GCGGGGATCAAATG
TCGTTTTG
252 RB-ATP-F2in CAGCGGTTGTCATT This study
TCTGGT
RB-ATP-R2in ACAAGTTCCAATGT
CTACCGT
412 RB-ATP-F1 ATGTCTGTTCCTGT Charoenwai
GAAGGAA et al. (2019)
RB-ATP-R2in ACAAGTTCCAATGT
CTACCGT
738 RB-ATP-F1 ATGTCTGTTCCTGT
GAAGGAA
RB-ATP-R1 TTATTCAACAACAG
CAATTGCG
MCP 460 RB-MC-F2in ACGATCTCACTGCA This study
CAGACT
RB-MC-R2in GGTGCCATCTGACA
CTGTTC
Tilapia lake virus Segment 250 7450/150R/ME2 TATCACGTGCGTAC Kembou Tsofack
(TiLV) 3 TCGTTCAGT et al. (2016)
ME1 GTTGGGCACAAGGC
ATCCTA
White spot VP28 141 VP28-qPCR-F AGGTGTGGAACAAC Mendoza-Cano
syndrome virus ACATCAAG and Sánchez-Paz
(WSSV) VP28-qPCR-R TGCCAACTTCATCC (2013)
TCATCA
530 Aquaculture International (2021) 29:527–538

Favogen Gel/PCR Purification Kit (Taiwan) prior to cloning into pGEM T-easy cloning vector
(Promega, USA). Sequences of respective inserted genes were confirmed by DNA sequencing
(Macrogen, South Korea). Quality and quantity of the recombinant plasmids were measured by
spectrophotometry at 260 and 280 nm. Plasmid copy number was calculated based on DNA
concentration and plasmid size using a web tool (https://cels.uri.edu/gsc/cndna.html).

Fish DNA template

DNA templates derived from clinical samples used in this study were from SDDV-infected Asian
sea bass (L. calcarifer). DNA previously extracted from spleen and liver of fish specimens during
mortality cases in 2016 and 2019 (Senapin et al. 2019; Sriisan et al. 2020) has been stored at −
20 °C. The former template contained SDDV DNA of 1.6 × 106 copies/μl, while the latter had
7.8 × 105 copies/μl that were quantified by SDDV quantitative PCR (Sriisan et al. 2020).

PCR conditions for 7 brands of Taq polymerase

Seven commercial brands of PCR test kits that employ Taq DNA polymerase enzyme were
used, namely, RBC Bioscience (Taiwan), Invitrogen (USA), PCRBiosystems (UK), Bio-Helix
(Taiwan), biotechrabbit (Germany), GeneAll (South Korea), and New England BioLabs
(USA). For convenient experimental practices, this study compared the tested polymerase
enzymes with similar setting conditions in which they might not be the optimal conditions of
each commercial enzyme we used here. The PCR reactions were carried out in 25 μl that
contained 0.5 μl of 10 μM forward primer, 0.5 μl of 10 μM reverse primer, 0.5 μl of 10 mM
dNTPs, 2 μl of plasmid DNA, and 0.125–0.5 units of Taq enzyme in 1X reaction buffer as
provided with each kit. Among the 7 brands, the final concentrations of MgCl2 ranged between
1.5 and 3.0 mM. PCR conditions were 94 °C for 5 min, followed by 30 cycles of 94 °C for
30 s, 62 °C for 30 s, and 68 °C (for BioLabs Taq polymerase) or 72 °C (for the 6 remaining
brands) for 30 s, followed by final heating at either 68 or 72 °C for 5 min. Biometra
TProfessional and BIORAD T100 Thermal Cyclers were used in this study. PCR products
were run on 1.5% agarose gels at 120 V and stained with ethidium bromide before visuali-
zation under UV light using a gel documentation system.

Testing reaction components

In this study, testing reaction components was carried out by 4 approaches. This included (i)
using 7 individual Taq enzymes in RBC Bioscience reaction buffer compared to their original
buffer, (ii) addition of a final concentration of 0.01% bovine serum albumin (BSA) (Catalog No.
B9001S, BioLabs), (iii) addition of a final concentration of 10 mM ammonium sulfate (Catalog
No. 1505193821, Ajax Finechem Pty Ltd.), and (iv) addition of 0.01% BSA and 10 mM
ammonium sulfate. Four Taq brands (RBC Bioscience, Invitrogen, biotechrabbit, and GeneAll)
were employed in the 3 latter experiments that were performed in duplicate. An appropriate set of
serial dilutions of tested plasmid control was used as template in PCR assays, i.e., 103 to 106
copies/μl for ATPase 738 bp, TiLV 250 bp and WSSV 141 bp amplifications, 103 to 105
copies/μl for ATPase 252 bp amplification, 102 to 105 copies/μl for ATPase 135 bp amplifica-
tion, and 104 to 107 copies/μl for MCP 460 bp amplification. Amplification performance of each
condition was evaluated by the detection sensitivity; i.e., the performance was indicated by the
lowest template dilution that can be detected.
Aquaculture International (2021) 29:527–538 531

SDDV PCR detection

DNA extracted from 2 individual SDDV-infected fish mentioned above were subjected to
semi-nested protocol as previously described (Charoenwai et al. 2019). Reactions were
performed using 2 μl of DNA template with (i) RBC Bioscience Taq polymerase, (ii)
biotechrabbit Taq polymerase, and (iii) biotechrabbit Taq polymerase plus 10 mM ammonium
sulfate. Expected band product sizes were 412 bp for low SDDV load and 412 bp together
with 738 bp for high viral load.

Results

Amplification of 6 target genes using 7 Taq polymerase enzymes

Seven different brands of Taq DNA polymerase enzymes, namely, RBC Bioscience,
Invitrogen, PCRBiosystems, Bio-Helix, biotechrabbit, GeneAll, and New England
BioLabs, were used for PCR amplification of 6 target viral genes including SDDV ATPase
738 bp, SDDV ATPase 252 bp, SDDV ATPase 135 bp, SDDV MCP 460 bp, TiLV
segment_3 250 bp, and WSSV VP28 141 bp. Performance of amplification was deter-
mined by the detection sensitivity using serially diluted respective plasmid as template.
The results summarized in Table 2 indicated that only RBC Bioscience, Invitrogen, and
Bio-Helix Taq polymerase brands can amplify SDDV ATPase 738 bp, and only the 2
former brands yielded SDDV ATPase 252 bp products, while other remaining enzymes
failed to amplify these targets. This problem was not observed in the amplification of other
tested SDDV regions (Table 2). For example, all of the enzymes can generate SDDV

Table 2 Amplification results of 6 target genes using 7 different brands of Taq polymerase enzymes

Primers Enzymes

RBC Invitrogen PCRBiosystems Bio- biotech GeneAll New England

Bioscience Helix rabbit BioLabs

SDDV ATPase ✓(105) ✓(105) X ✓(106) X X X

738 bp
ATPase ✓(103) ✓(104) X X X X X
252 bp*
ATPase ✓(103) ✓(103) ✓(103) ✓(103) ✓(103) ✓(103) ✓(103)
135 bp
MCP ✓(104) ✓(104) ✓(105) ✓(105) ✓(104) ✓(107) ✓(105)
460 bp
TiLV segment_3 ✓(103) ✓(103) ✓(103) ✓(103) ✓(103) ✓(104) ✓(104)
250 bp
WSSV VP28 ✓(104) ✓(104) ✓(105) ✓(105) ✓(105) ✓(104) ✓(105)
141 bp

Primers target SDDV ATPase (738, 252, and 135 bp) and MCP (460 bp) coding genes as well as TiLV
segment_3 and WSSV VP28 partial fragments. Serial dilution solutions of plasmid containing respective target
genes were used as template. Numbers in brackets represent the detection limit (in plasmid copies per microliter)
of each amplification
✓, product obtained; X, no product obtained; *, results from duplicate tests, gray highlights indicated represen-
tative gel results shown in Fig. 1a–b
532 Aquaculture International (2021) 29:527–538

ATPase 135 bp with the same detection limits of 103, whereas SDDV MCP 460 bp could
be amplified by all 7 enzymes with sensitivity ranging from 104 to 107 copies (Table 2).
TiLV segment_3 250 bp and WSSV VP28 141 bp band products could be produced by all
tested Taq polymerase enzymes with detection limits ranging from 103 to 104 copies and
104 to 105 copies, respectively (Table 2). Representative agarose gels of PCR results from
WSSV VP28 141 bp and SDDV ATPase 252 bp amplifications are shown in Fig. 1 a and b,
respectively. Note that SDDV ATPase 252 bp PCR was run in duplicate, and one of the
tests is shown in Fig. 1b.

Fig. 1 Representative agarose gels of PCR results obtained using 7 different brands of Taq polymerase enzymes
with primers targeting (a) WSSV VP28 141 bp and (b) SDDV ATPase 252 bp. (c) Amplification results of
SDDV ATPase 252 bp products using 6 brands of Taq polymerase enzymes in RBC Bioscience reaction buffer.
Serial dilution solutions of plasmid containing respective target genes were used as template. Underlines mark the
detection limit of each amplification. M 2-log DNA marker (New England Biolabs), N no template control
Aquaculture International (2021) 29:527–538 533

Use of RBC bioscience reaction buffer improved detection sensitivity

The above findings led us to hypothesize that reaction components had effects to amplification
performance of SDDV ATPase 738 bp and 252 bp. To prove this, PCR reactions of SDDV
ATPase 252 bp were conducted using 7 different Taq polymerase enzymes, but their respective
buffer was replaced by buffer from RBC Bioscience. The results shown in Fig. 1c demonstrated
that the 5 commercial enzymes (namely, PCRBiosystems, Bio-Helix, biotechrabbit, GeneAll, and
New England BioLabs) that did not previously amplify SDDV ATPase 252 bp products could
yield the products with sensitivity of 103 to 104 copies. Reactions using enzyme from Invitrogen
with buffer from RBC Bioscience had 10 times better sensitivity, i.e., from 104 being improved to
103 copies (Fig. 1c). When tested using the same condition modification with 3 other represen-
tative target genes (SDDV MCP 460 bp, TiLV Segment_3 250 bp, and WSSV VP28 141 bp),
detection sensitivity remained the same for some assays but increased in most of the reactions
from 10 to 1000 times (Table 3). For instance, detection sensitivity of WSSV VP28 141 bp was
increased 10 times (105 becoming 104 copies) for all 7 enzymes that were run in reaction buffer
from RBC Bioscience. The 1000 times increased sensitivity was observed for enzyme brands
GeneAll and New England BioLabs in amplification of SDDV MCP 460 bp, i.e., from 107 being
improved to 104 copies (Table 3). The findings implied that reaction components in RBC
Bioscience had effects to amplification performance in most of the reactions.

Effect of addition of BSA and ammonium sulfate to detection sensitivity

When reaction components were compared among 7 commercial brands of Taq polymerase
enzymes, there were ammonium sulfate and bovine serum albumin (BSA) in RBC Bioscience
buffer that were not present or specified in other enzyme brands. To test whether these 2
components had effect to amplification performance, PCR reactions of SDDV ATPase 252 bp
were conducted using 4 selected commercial Taq polymerase brands (RBC Bioscience,
Invitrogen, biotechrabbit, and GeneAll) in (i) its original reaction buffer, (ii) original buffer
plus 0.01% BSA, (iii) original buffer plus 10 mM ammonium sulfate, and (iv) original buffer
plus 0.01% BSA and 10 mM ammonium sulfate. Experiments were performed in duplicate.
The results shown in Table 4 revealed that addition of ammonium sulfate (iii) could yield the
252 bp products from biotechrabbit and GeneAll Taq enzymes that previously failed to amplify
and increased 10 times sensitivity of Invitrogen polymerase in one of the replicates. Detection
sensitivity of enzyme from RBC Bioscience remained the same in this ammonium sulfate
addition condition (Table 4). Addition of BSA (ii) reduced RBC Bioscience enzyme efficiency
and gave no improvement to both replicate tests of Invitrogen and biotechrabbit enzymes and
one replicate of GeneAll polymerase. Reactions with both BSA and ammonium sulfate added
(iv) did not improve efficiency to most of the reactions tested, except for GeneAll enzyme and
one replicate from biotechrabbit polymerase. Taken together, the findings suggested that
ammonium sulfate supplement in reaction buffer alone can improve PCR detection sensitivity.
Figure 2 demonstrates representative PCR results from one of the replicate tests.

Application of adding ammonium sulfate in SDDV PCR detection

In this study, Taq DNA polymerase enzymes from RBC Bioscience and biotechrabbit were
used in SDDV PCR detection using semi-nested protocol as previously published
(Charoenwai et al. 2019). Serial dilutions of fish DNA extracted from 2 clinical samples
 534

Table 3 Amplification results of 4 target genes using 7 different brands of Taq polymerase enzymes in their original buffer compared to those in reaction buffer of RBC Bioscience
brand. Indicated numbers represent the detection limit (copies/μl) of each amplification

Virus & Combination

gene target
Enzyme RBC Invitrogen Invitrogen PCRBio PCRBio Bio- Bio-Helix biotech biotech GeneAll GeneAll New New
Bioscience systems systems Helix rabbit rabbit England England
BioLabs BioLabs
Buffer RBC Invitrogen RBC PCRBio RBC Bio- RBC biotech RBC GeneAll RBC New RBC
Bioscience Bioscience systems Bioscience Helix Bioscience rabbit Bioscience Bioscience England Bioscience
BioLabs

SDDV ATPase 103 104 103 X 103 X 104 X 104 X 103 X 103
252 bp
SDDV MCP 460 bp 104 104 104 105 104 105 105 104 104 107 104 107 104
TiLV Segment_3 103 103 103 103 103 103 103 103 103 104 103 104 103
250 bp
WSSV VP28 141 bp 105 105 104 105 104 105 104 105 104 105 104 105 104

X, no product obtained, gray highlights indicated representative gel results shown in Fig. 1c
Aquaculture International (2021) 29:527–538
Aquaculture International (2021) 29:527–538 535

Table 4 Amplification results of SDDV ATPase 252 bp products using 4 different brands of Taq polymerase
enzymes in 4 conditions

Conditions Enzymes

RBC Bioscience Invitrogen biotechrabbit GeneAll

Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2 Rep 1 Rep 2

Original buffer ✓ (103) ✓ (103) ✓ (104) ✓ (104) X X X X

+ BSA ✓ (104) ✓ (104) X X X X X ✓(105)
+ Ammonium sulfate ✓ (103) ✓ (103) ✓ (103) ✓ (104) ✓ (104) ✓ (103) ✓(104) ✓(104)
+ BSA & Ammonium sulfate ✓ (104) ✓ (105) X X ✓ (105) X ✓ (103) ✓ (105)

Experiments were performed in duplicate. Numbers in brackets represent the detection limit (copies//μl) of each
amplification
Gray highlights indicated representative gel results shown in Fig. 2. ✓, product obtained; X, no product obtained;
Rep, replicate

with known SDDV loads were used as template. The results shown in Fig. 3 demonstrated
that, when using their original buffer, RBC Bioscience Taq enzyme can detect as few as 160
SDDV copies and 780 copies in diluted fish samples 1 and 2, respectively, while
biotechrabbit enzyme yielded positive results from only template dilutions with relatively
high viral loads (≥ 1.6 × 104 and 7.8 × 104 SDDV copies). However, detection sensitivity of
biotechrabbit Taq polymerase was 100 times increased when ammonium sulfate was added
in the reactions in which the efficiency became the same as that of RBC Bioscience Taq
enzyme (Fig. 3).

Fig. 2 Representative amplification results of SDDV ATPase 252 bp products using 4 different brands of Taq
polymerase with 4 conditions; original buffer, addition of BSA, addition of ammonium sulfate, and BSA +
ammonium sulfate. Serial dilution solutions of plasmid control were used as template. Underlines mark the
detection limit of each amplification. M 2-log DNA marker (New England Biolabs), N no template control
536 Aquaculture International (2021) 29:527–538

Fig. 3 SDDV detection assay using semi-nested PCR protocol. Taq polymerase enzymes from RBC Bioscience
and biotechrabbit with their respective buffer were used. Reactions using biotechrabbit Taq polymerase added
with 10 mM ammonium sulfate were also performed. Serial dilution solutions of DNA extracted from 2
individual SDDV-infected fish with known SDDV DNA load were used as template. M 2-log DNA marker
(New England Biolabs), P positive control plasmid, N no template control

Discussion

Polymerase chain reaction (PCR) has been applied for diagnostics of infectious pathogens not
only in human, plants, and terrestrial animals but also aquatic organisms worldwide (Adams
and Thompson 2011). Nowadays, there are a variety of commercial brands providing en-
zymes, reaction buffers, and dNTPs for molecular laboratories, and their detailed components
sometimes differ. The present study revealed that in some established PCR protocols, changing
of Taq DNA polymerase brand (together with reaction buffer provided) affected seriously to
results in term of sensitivity (or amplification efficiency). The present study revealed that all
tested commercial Taq enzymes had comparable amplification efficiency in most of the gene
targets assayed. However, the change of Taq enzyme brands greatly affected amplifications of
SDDV ATPase 738 and 252 bp target products. Subsequently, it was found that presence of
ammonium sulfate (10 mM) in PCR reactions improved amplification sensitivity when
compared with the reaction buffer without this component. From literature review, it has been
known that ammonium ion (NH4+) stabilizes DNA polymerase and dNTPs, eliminates PCR
inhibitors, solves secondary structure of nucleic acid making it more accessible to reaction, and
enhances specificity between primer-template base-pairing (Park and Kim 1996; Korfhage
et al. 2002; Pelt-Verkuil et al. 2008).
In the original published semi-nested PCR protocol (Charoenwai et al. 2019) for detection
of SDDV in Asian sea bass, RBC Bioscience Taq polymerase was used, and the reaction
buffer already contained 10 mM ammonium sulfate. Its detection limit was reported to be 100
SDDV DNA copies/μl (Charoenwai et al. 2019). However, when the protocol was modified
by changing Taq DNA polymerase brand and buffer without ammonium sulfate, detection
sensitivity was considerably reduced. Detection sensitivity was regained the same as original
protocol when supplemented with this component into the reaction. We thus recommend that
those who use our semi-nested SDDV PCR protocol (Charoenwai et al. 2019), without access
to the Taq DNA polymerase brand described in the original protocol, modification should be
considered by using an alternative brand that contains ammonium sulfate in its reaction buffer
Aquaculture International (2021) 29:527–538 537

or supplement this component into the PCR reaction to avoid false negative detection result.
This approach might also be considered a solution to troubleshoot amplification problem that
occurred with other PCR protocols in molecular diagnostic laboratories.

Funding This project was supported by a research grant from Mahidol University (grant number 22531).

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

Ethics approval Not applicable.

References

Adams A, Thompson KD (2011) Development of diagnostics for aquaculture: challenges and opportunities.
Aqua Res 42:93–102
Charoenwai O, Meemetta W, Sonthi M, Dong HT, Senapin S (2019) A validated semi-nested PCR for rapid
detection of scale drop disease virus (SDDV) in Asian sea bass (Lates calcarifer). J Virol Methods 268:37–41
de Groof A, Guelen L, Deijs M, Van Der Wal Y, Miyata M, Ng KS, Van Grinsven L, Simmelink B, Biermann
Y, Grisez L, Van Lent J, De Ronde A, Chang SF, Schrier C, Van Der Hoek L (2015) A novel virus causes
scale drop disease in Lates calcarifer. PLoS Pathog 11:e1005074
Dong HT, Ataguba GA, Khunrae P, Rattanarojpong T, Senapin S (2017) Evidence of TiLV infection in tilapia
hatcheries from 2012 to 2017 reveals probable global spread of the disease. Aquaculture 479:579–583
Eyngor M, Zamostiano R, Kembou Tsofack JE, Berkowitz A, Bercovier H, Tinman S, Lev M, Hurvitz A,
Galeotti M, Bacharach E, Eldar A (2014) Identification of a novel RNA virus lethal to tilapia. J Clin
Microbiol 52:4137–4146
Ferguson HW, Kabuusu R, Beltran S, Reyes E, Lince JA, del Pozo J (2014) Syncytial hepatitis of farmed tilapia,
Oreochromis niloticus (L.): a case report. J Fish Dis 37:583–589
Gibson-Kueh S, Chee D, Chen J, Wang YH, Tay S, Leong LN, Ng ML, Jones JB, Nicholls PK, Ferguson HW
(2012) The pathology of “scale drop syndrome” in Asian seabass, Lates calcarifer Bloch, a first description.
J Fish Dis 35:19–27
Jansen MD, Dong HT, Mohan CV (2018) Tilapia lake virus: a threat to the global tilapia industry? Rev Aquacult
11:725–739. https://doi.org/10.1111/raq.12254
Kembou Tsofack JE, Zamostiano R, Watted S, Berkowitz A, Rosenbluth E, Mishra N, Briese T, Lipkin WI,
Kabuusu RM, Ferguson H, Del Pozo J, Eldar A, Bacharach E (2016) Detection of Tilapia Lake virus (TiLV)
in clinical samples by culturing and nested RT-PCR. J Clin Microbiol 55:759
Korfhage C, Oelmüller U, Wyrich R (2002) Ammonium sulfate for neutralization of inhibitory effects.
EP1356122A2 (European Patent) https://patents.google.com/patent/EP1356122A2/en
Machimbirike VI, Jansen MD, Senapin S, Khunrae P, Rattanarojpong T, Dong HT (2019) Viral infections in
tilapines: more than just tilapia lake virus. Aquaculture 503:508–518
Mendoza-Cano F, Sánchez-Paz A (2013) Development and validation of a quantitative real-time polymerase
chain assay for universal detection of the white spot syndrome virus in marine crustaceans. Virol J 10:186
Nurliyana M, Lukman B, Ina-Salwany MY, Zamri-Saad M, Annas S, Dong HT, Rodkhum C, Amal MNA
(2020) First evidence of scale drop disease virus in farmed Asian seabass (Lates calcarifer) in Malaysia.
Aquaculture 528:735600
Park HO, Kim JJ (1996) Lyophilized reagent for polymerase chain reaction. US5861251A (US patent) https://
patents.google.com/patent/US5861251A/en
Pelt-Verkuil EV, Belkum AV, Hays JP (2008) Principles and technical aspects of PCR amplification. Springer,
Dordrecht
Sangsuriya P, Huang JY, Chu YF, Phiwsaiya K, Leekitcharoenphon P, Meemetta W, Senapin S, Huang WP,
Withyachumnarnkul B, Flegel TW, Lo CF (2014) Construction and application of a protein interaction map
for white spot syndrome virus (WSSV). Mol Cell Proteomics 13:269–282
Senapin S, Dong HT, Meemetta W, Gangnonngiw W, Sangsuriya P, Vanichviriyakit R, Sonthi M, Nuangsaeng B
(2019) Mortality from scale drop disease in farmed Lates calcarifer in Southeast Asia. J. Fish Dis 42: 119–127
538 Aquaculture International (2021) 29:527–538

Sriisan S, Boonchird C, Thitamadee S, Sonthi M, Dong HT, Senapin S (2020) A sensitive and specific SYBR
green-based qPCR assay for detecting scale drop disease virus (SDDV) in Asian sea bass. Dis Aquat Org
139:131–137
Thitamadee S, Prachumwat A, Srisala J, Jaroenlak P, Salachan PV, Sritunyalucksana K, Flegel TW,
Itsathitphaisarn O (2016) Review of current disease threats for cultivated penaeid shrimp in Asia.
Aquaculture 452:69–87

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations.