Journal of Invertebrate Pathology xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Journal of Invertebrate Pathology

journal homepage: www.elsevier.com/locate/jip

Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan

probe real-time PCR
Ya-Mei Liua,b,1, Liang Qiua,b,1, An-Zhi Shenga, Xiao-Yuan Wana, Dong-Yuan Chenga,b,
⁎
Jie Huanga,b,
a
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of
Maricultural Organism Disease Control, Ministry of Agriculture, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Yellow Sea Fisheries Research
Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China
b
Shanghai Ocean University, Shanghai 201306, China

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

Keywords: A TaqMan probe and a pair of specific primers were selected from the small subunit ribosomal DNA (SSU rDNA)
TaqMan-based qPCR sequence of Enterocytozoon hepatopenaei (EHP); this real-time PCR assay was developed and optimized. It showed
Enterocytozoon hepatopenaei (EHP) a good linearity in detecting standards of EHP SSU rDNA fragments from 4 × 102 to 4 × 108 copies/reaction
Body mass index (BMI) using the established method. The detection limit of the qPCR method was as low as 4 × 101 copies per reaction,
which was higher than the conventional PCR and SYBR Green I-based EHP qPCR reported. Using the qPCR assay,
EHP was detected in four batches of slow-growing Penaeus vannamei specimens collected from Tianjin and
Zhejiang Province in China was detected using qPCR. The results showed that all the hepatopancreas from the
slow-growing P. vannamei specimens were detected as EHP-positive. EHP copies of hepatopancreas in some
batches had a negative correlation with the body mass index (BMI) of shrimps; however, not all batches of
specimens had this negative correlation between EHP copies of hepatopancreas and BMI. This qPCR technique is
sensitive, specific and easy to perform (96 tests in < 3 h), which provides technical support for the detection and
prevention of EHP.

1. Introduction this disease is mainly through histological observation, PCR assay

In recent years, the problem of slow growth in shrimp has increas-
ingly been reported in shrimp farms in Southeast Asia countries. The
shrimp cultured in these farms were severely infected with a micro-
sporidian, namely Enterocytozoon hepatopenaei (EHP), which was first
found in slow-growing Penaeus monodon cultured in Thailand (Tourtip
et al., 2009). As with the outbreak of acute hepatopancreas necrosis
disease (AHPND), the EHP infection was not given enough attention in
the early stages (Tangprasittipap et al., 2013). Thus EHP widely spread
in cultured P. monodon and P. vannamei, the problem of slow growth in
shrimp has become more and more common and the economic loss has
become increasingly serious, thus it threatens the development of the
whole shrimp aquaculture directly or indirectly (Tang et al., 2015).
EHP, an intracellular parasite, belongs to the family Nosematidae, the
genus Enterocytozoon and replicate in the cytoplasm of epithelial cells in
the hepatopancreas of the host (Tang et al., 2015). No obvious clinical
symptoms have been observed in EHP infection and the diagnosis of
(Tangprasittipap et al., 2013), in situ hybridization assay with digox-
igenin-labeled probe, and loop-mediated isothermal amplification de-
tection assay (Amornrat et al., 2013; Suebsing et al., 2013; Tourtip
et al., 2009). However, these detection assays could only qualitatively
describe the pathogenic microorganism without carrying out quanti-
tative research.
A SYBR Green I based qPCR assay was established in our laboratory
to detect EHP in former research (Liu et al., 2016). This method pro-
vides a useful technique to support the detection and research for
controlling EHP with relatively higher sensitivity and by a quantitative
method. Among the quantitative detection methods, TaqMan probe-
based qPCR assay uses a specific probe which can hybridize with the
target sequence with a high specificity. Therefore, the TaqMan probe-
based qPCR assay could effectively prevent influences caused by non-
specific products in the PCR and further improve the accuracy of
quantitative assay (Yue et al., 2006), and has been extensively applied
in the detection of aquatic animal pathogens. In the Office International

⁎
Corresponding author at: Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, No.
106, Nanjing Road, Shinan District, Qingdao 266071, China.
E-mail address: huangjie@ysfri.ac.cn (J. Huang).
1
These authors contributed to the work equally and should be regarded as co-first authors.

https://doi.org/10.1016/j.jip.2017.12.006
Received 23 June 2017; Received in revised form 13 December 2017; Accepted 21 December 2017
0022-2011/ © 2017 Elsevier Inc. All rights reserved.

Please cite this article as: Liu, Y.-M., Journal of Invertebrate Pathology (2017), https://doi.org/10.1016/j.jip.2017.12.006
Y.-M. Liu et al.
Des Epizooties (OIE) aquatic animal diagnosis standard of shrimp pa-
thogens, the TaqMan probe assay often becomes the standard method
for quantitative detection including the following 12 pathogens: Ba-
trachochytrium dendrobatidis, infectious hypodermal and hematopoietic
necrosis virus (IHHNV), infectious myonecrosis virus (IMNV), necro-
tizing hepatopancreatitis bacterium (NHPB), shrimp TSV, shrimp white
spot syndrome virus (WSSV), infectious salmon anemia virus (ISAV),
koi herpes virus (KHV), viral haemorrhagic septicemia virus (VHSV),
striped jack nervous necrosis virus (SJNNV), abalone herpes (AbHV),
and oysters herpes (OsHV-1) (OIE, 2015).
To quantitatively detect EHP more accurately, a TaqMan probe
qPCR detection technique was established in this study and was used
for detection in farm samples.
2. Materials and methods
2.1. Animal samples
Farm samples in this experiment used four batches of P. vannamei in
a total of 188 individuals collected from farms in Tianjin and Zhejiang
Province in China in 2015. The samples collected from Tianjin were
coded 20150710101–20150710106, 20150710201–20150710220
(Farm 1) and 20150811001–20150811100 (Farm 2), and samples from
Zhejiang were coded 20150920101–20150920162. The body length
range was 3.3–9.2 cm and the weight range was 0.21–3.85 g. The he-
patopancreas tissues of shrimp were taken after dissection and pre-
served separately using 95% ethanol with more than three times the
volume.
2.2. Extraction of hepatopancreas DNA
After removal of ethanol by rinsing with sterile water, 30 mg of
hepatopancreas tissue from P. vannamei preserved in 95% ethanol was
taken to extract DNA using a TIANamp Marine Animals DNA Kit
(Tiangen Biotech, Beijing, China). The extracted HpDNA was diluted to
50 ng/µL after determination of DNA concentration with a nucleic acid
analyzer (Nanodrop 2000c, Thermo Fisher Scientific, Waltham, MA,
USA), and then stored at −20 °C.
2.3. EHP primer/probe design and conventional PCR amplification
A pair of specific primers, F157 (5′-AGT AAA CTA TGC CGA CAA-3′)
and R157 (5′-AAT TAA GCA GCA CAA TCC-3′), and a TaqMan probe
(5′-FAM-TCC TGG TAG TGT CCT TCC GT-TAMRA-3′) were designed
based on the SSU rDNA sequence of EHP published in GenBank
(KF362129) using AlleleID version 5.01 (PREMIER Biosoft, Palo Alto,
California, USA). The length of the target fragment was expected to be
157 bp. The conventional PCR amplification system in 25 µL contained
12.5 µL 2× PCR Mix (TaKaRa, Dalian, China), 0.5 µL F157 (10 μmol/L)
and 0.5 µL R157 (10 μmol/L). The amplification was initiated by de-
naturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 20 s,
54 °C for 30 s and 72 °C for 30 s, and a final extension at 72 °C for
10 min. PCR products (5 μL) were separated and analyzed in 1%
agarose gels containing GeneFinder (Bio-V, Shanghai, China).
2.4. Preparation of a quantitative standard
DNA from EHP-infected samples was used as the template, and the
specific primers F157 and R157 described earlier were used to perform
conventional PCR amplification to obtain the target products. Then the
products was cloned in E. coli DH5α using a pMD18-T vector and ver-
ified by sequencing (Sangon, Shanghai, China). The recombinant
plasmid was extracted from the clone with a Mini Plasmid Extraction
Kit (Sangon Biotech, Shanghai, China) and determined with a nucleic
acid analyzer as 87.6 ng/µL which was equal to 4 × 1010 copies/µL.
The recombinant plasmid was used as the quantitative standard and
2
Journal of Invertebrate Pathology xxx (xxxx) xxx–xxx
stored at −80 °C.
2.5. The establishment and optimization of the TaqMan-based EHP qPCR
assay
The reaction mixture of TaqMan probe-based quantitative PCR for
EHP (TaqMan-based EHP qPCR) in 25 μL was prepared according to the
manufacturer’s instructions of Premix Ex Taq™ (Probe qPCR, TaKaRa).
The concentration of primers and probe was optimized using a 5 × 5
matrix method, by which the final concentration gradients of the pri-
mers were, respectively, 0.1, 0.2, 0.3, 0.4, and 0.5 μM, those of the
TaqMan probe were, respectively, 0.05, 0.1, 0.15, 0.2, and 0.25 μM, and
the other components included 2× Premix Ex Taq™ 12.5 µL, DNA
template 1 μL, and nuclease-free water was added to make a volume of
25 μL. The amplification reaction was performed in a qPCR instrument
CFX96 (BIO-Rad, Hercules, California, USA) and the reaction procedure
was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for
30 s.
2.6. Construction of a standard curve of TaqMan-based EHP qPCR
The plasmid standard containing an EHP target fragment was di-
luted in a 10-fold series to 11 gradients (4 × 1010–4 × 100 copies).
Three parallels of each dilution were used as the template of TaqMan
qPCR assays in which the above-optimized conditions were applied to
construct a standard curve of copy numbers of EHP plasmid standards
to cycle threshold (Ct) values and to analyze the correlation coefficient,
as well as amplification efficiency.
2.7. Analytic specificity of TaqMan-based EHP qPCR
To test the analytic specificity of TaqMan-based EHP qPCR, we used
DNA extracted from shrimp tissue infected with different pathogens as
the template in the qPCR assays, including DNA of P. vannamei infected
with IHHNV, hepatopancreatic parvovirus, WSSV, Vibrio para-
haemolyticus, which cause acute hepatopancreas necrosis disease
(VPAHPND) and cDNA reverse transcribed from the RNA of shrimp tissue
infected with covert mortality nodavirus (CMNV) and TSV. The nega-
tive, positive, and blank controls were the DNA of healthy P. vannamei,
DNA of P. vannamei infected with EHP, and water, respectively.
2.8. Comparative diagnostic sensitivity of TaqMan-based EHP qPCR with
nested PCR and SBYR Green I qPCR
Sixty-two DNA samples extracted from hepatopancreas of P. van-
namei collected in Zhoushan and Zhejiang Province with unknown
status of EHP infection were tested by TaqMan-based EHP qPCR, nested
PCR (Amornrat et al., 2013) and SBYR Green I qPCR method (Liu et al.,
2016). Diagnostic sensitivity and diagnostic specificity were calculated
according to the OIE manual (OIE, 2016).
2.9. Correlation between the EHP loads in hepatopancreas and growth of
shrimp
P. vannamei populations with obvious growth differences were col-
lected from two farms in Tianjin and one farm in Zhejiang Province in
China. For Farm 1 in Tianjin Province, samples were collected before
and after a week of tentative medication by mixing well with the daily
shrimp feeds to treat EHP. Body weight (W, g) and biological body
length (L, cm) of each individual in the populations were measured to
calculate body mass index (BMI, BMI = W/L2 × 10, kg/m2). DNA ex-
tracted from hepatopancreas were detected through TaqMan-based
EHP qPCR assay, and correlation of EHP loads and growth was drawn
according to the BMI of P. vannamei. With Log(EHP copies/ng HpDNA)
and BMI as the Y and X value, respectively, scatter diagram and cor-
relation analysis was carried out using Office Excel (Microsoft,
Y.-M. Liu et al. Journal of Invertebrate Pathology xxx (xxxx) xxx–xxx

Table 1 3.4. Sensitivity of TaqMan-based EHP qPCR

The BLAST results for the designed TaqMan qPCR primers and probe.
Species
Enterocytozoon hepatopenaei
(×20)
Enterospora nucleophile (×6)
Enterocytozoon bieneusi (×40)
Enterocytozoon bieneusi (×7)
Fluorescent signals were observed when the standard template used
Difference with Difference with probe
primers in the TaqMan-based EHP qPCR ranged from 4 × 101 to 4 × 108 copies
after 40 cycles of amplification (Fig. 1B). When the template amount
0 0 was 4 × 100 copies, fluorescence was detected in two of the six paral-
lels after 40 cycles of amplification. The results indicated that a tem-
1 base 0
1 base 0 plate of at least 4 × 101 copies could be detected by TaqMan-based EHP
2 bases 0 qPCR and the method reached a higher sensitivity.

GenBank Accession Number: 20 isolates of Enterocytozoon hepatopenaei
(MF977742–MF977746, KX981865, KX688794, MF417474, KY643648, KX932044,
KX932043, KY593127, KY075856, KX013492, KX013491, KU179095, KP759285,
KF362130, KF362129 and FJ496356); 6 isolates of Enterospora nucleophile
(KF135641–KF135645 and JX101917); 40 isolates of Enterocytozoon bieneusi (KJ019869,
AH012971, KF305580, KF305579, KJ475400, KF271: 486, 488, 489, 491, 493–495,
497–510, 512, 513, 515, 518, 519, JQ769381, JN107808, DQ793212, AY257180,
AY298728, AF023245, AF024657, L16868 and L07123); 7 isolates of Enterocytozoon bi-
eneusi (KJ719491, KF271517, KF271516, KF271496, KF271492, KF271490 and
KF271487).
Redmond, CA, USA).
3.5. Standard curve between cycle threshold of TaqMan-based EHP qPCR
and starting quantity of template
The standard curve between amplification cycle threshold (Ct) of
TaqMan-based EHP qPCR and starting quantity (SQ) of template was
constructed with serial dilutions ranging from 4 × 100 to 4 × 108 co-
pies of standard template of EHP. The corresponding relation between
Ct and logarithmic SQ showed a good linear correlation when SQ was
within the range of 4 × 102–4 × 108 copies (Fig. 1C): Ct = −3.621 log
(SQ) + 45.180, correlation coefficient R2 = 0.995 and the amplifica-
tion efficiency was 88.9%.

3.6. Repeatability analysis result of TaqMan-based EHP qPCR detection

3. Results
assay
3.1. Design of TaqMan-based EHP qPCR primers
Standard templates of EHP with seven concentrations ranged from
4 × 101 to 4 × 107 copies were selected to calculate the mean value,
The target fragment sequences of designed primers F157 and R157
standard deviation (SD) and coefficient of variation (CV) of the Ct value
were submitted to the BLAST tool in NCBI to search for any related
(Table 2). The result showed that Ct values of the three intra-assay
sequences. A total of 73 nucleotide sequences of the small subunit ri-
parallels were basically consistent while CV < 1%, indicating that the
bosomal RNA (SSU rRNA) genes from EHP-related microsporidian taxa
intra-assay repeatability were reliable.
were retrieved from the Gen-Bank database and compared with the
Standards with seven concentrations that ranged from 4 × 101 to
primers and probe (Table 1). The primers F157 and R157 showed no
4 × 107 copies stored at −20 °C were selected for the inter-assay re-
difference with 20 isolates of Enterocytozoon hepatopenaei, 1 nucleotide
peatability analysis (Table 3). The amplification data of standards di-
with 6 isolates of Enterospora nucleophile, 1 base with 40 isolates of
luted and stored for 40 days was compared and independent sample t-
Enterocytozoon bieneusi and 2 bases with 7 isolates of Enterocytozoon
test was carried out using SPSS statistical software (IBM, Armonk, New
bieneusi. The probe showed no difference with all 73 species.
York, USA). The results showed that the P-value of the seven con-
centrations was larger than .05, indicating there was no significant
difference in the standard curve between the standards stored at
3.2. Establishment of TaqMan-based EHP qPCR amplification conditions
−20 °C for 40 days and just diluted. Result showed that the developed
assay had good repeatability.
The amplification of TaqMan-based EHP qPCR was conducted using
4 × 105 copies of the recombinant plasmid DNA containing a target
3.7. Comparative diagnostic sensitivity of TaqMan qPCR with nested PCR
fragment of EHP SSU rRNA as the standard template. The results
in farm sample detection
showed that the optimum concentration of primers F157/R157 was
0.4 μM, and that of the TaqMan probe was 0.25 μM in the TaqMan-
Sixty-two individuals of P. vannamei that have obvious shorter body
based EHP qPCR amplification mixture. So, the optimized TaqMan-
than other individuals in the same pond were collected in one shrimp
based EHP qPCR mixture in 25 µL contains 2× Premix Ex Taq™ 12.5 µL,
farm in Zhoushan, Zhejiang Province, and were detected simulta-
10 μM primers F157 and R157 1 µL each, 10 μM TaqMan probe
neously by TaqMan-based EHP qPCR, nested PCR (Amornrat et al.,
0.625 µL, and 50 ng of DNA template.
2013) and SYBR Green I qPCR-EHP (Liu et al., 2016). Four negative and
58 positive samples were determined by TaqMan-based EHP qPCR with
a positive detection rate of 93.5%, and the quantity of EHP SSU rDNA
3.3. Specificity of TaqMan-based EHP qPCR
was 6.54 × 101–3.90 × 105 copies/µL after conversion according to the
Ct value. Fourteen negative and 48 positive samples were detected by
The specificity of TaqMan-based EHP qPCR was tested with tissue
nested PCR with a positive detection rate of 77.4%. Six negative and 56
samples infected with different shrimp pathogens, including DNA from
positive samples were detected by SYBR Green I qPCR-EHP with a
P. vannamei infected with IHHNV, DNA from F. chinensis infected with
positive detection rate of 90.3%, and EHP SSU rDNA was
hepatopancreatic parvovirus, DNA from purified WSSV, DNA from P.
7.89 × 101–1.13 × 105 copies/µL after conversion according to the Ct
vannamei infected with V. parahaemolyticus, cDNA of RNA from P.
value. From the result comparison of the three detection assays, 10
vannamei infected with CMNV, cDNA of RNA from P. vannamei infected
positive samples detected with TaqMan-based EHP qPCR were de-
with TSV, under the foregoing optimized qPCR conditions.
termined negative by nested PCR, and three positive samples detected
Amplification curves showed that a fluorescent signal of amplification
using TaqMan-based EHP qPCR were determined negative with SYBR
only existed in the sample demonstrated EHP-positive by conventional
Green I qPCR-EHP (Fig. 2). The difference among these detection re-
PCR, while no detectable fluorescent signal existed in the other samples
sults correlated to SSU rDNA copies of EHP detected in the samples
and negative control (Fig. 1A), indicating that the method had good
through TaqMan-based EHP qPCR. When the SSU rDNA copies of EHP
specificity.
in the DNA samples detected by TaqMan-based EHP qPCR ranged from

3
Y.-M. Liu et al. Journal of Invertebrate Pathology xxx (xxxx) xxx–xxx

Fig. 1. A: Amplification curve of specificity analysis of the TaqMan qPCR for the SSU rDNA of microsporidian E. hepatopenaei. B: Amplification curve of TaqMan probe qPCR for E.
hepatopenaei SSU rDNA with plasmid standard templates. C: The standard curve of SSU rDNA TaqMan qPCR for microsporidian E. hepatopenaei.

Table 2
Intra-assay variability of TaqMan qPCR for E. hepatopenaei.

Template (copies) Mean Ct of Intra-assay SD CV

4 × 107 17.72 0.0296 0.17%

4 × 106 21.26 0.1116 0.53%
4 × 105 24.23 0.0980 0.40%
4 × 104 27.71 0.0852 0.31%
4 × 103 30.83 0.3832 0.95%
4 × 102 33.95 0.2823 0.83%
4 × 101 38.73 0.4253 0.90%

Table 3
Inter-assay variability of TaqMan qPCR for E. hepatopenaei.

Template (copies) P-value

7
4 × 10 .326
4 × 106 .172 Fig. 2. Comparison between nested PCR and qPCR for E. hepatopenaei using cultured
4 × 105 .690 shrimp samples.
4 × 104 .053
4 × 103 .320
4 × 102 .222 TaqMan-based EHP qPCR was (6.33 ± 0.99) × 101 copies/µL, in-
4 × 101 .231 dicating that in the detection of farm samples, the diagnostic sensitivity
of TaqMan-based EHP qPCR was higher than nested PCR and SYBR
Green I qPCR by 7.6 ± 4.2 times and 1.3 ± 0.2, respectively. The
(4.48 ± 0.54) × 102 to (4.98 ± 3.69) × 102 copies/µL, the reprodu- ratio of log value of TaqMan-based EHP qPCR and SYBR Green I qPCR-
cibility of positive or negative attributes of nested PCR detection was EHP was 1.026 ± 0.119. There was highly correlate relations between
unstable. The sample was determined negative by SYBR Green I qPCR the two methods and the correlation coefficient R was 0.8978.
when the SSU rDNA copies of EHP ranged from (6.33 ± 0.99) × 101 to
(6.50 ± 0.20) × 101 copies/µL and was (4.98 ± 3.69) × 102 copies/
µL. The lowest SSU rDNA copies of EHP detected in the samples by

4
Y.-M. Liu et al.
Table 4
Results of tests by TaqMan-based EHP qPCR, nested PCR, and SYBR green I qPCR using farm samples.
Detection assay Detection result TaqMan-based EHP qPCR
Positive (58) Negative (4)
Nested PCR Positive 48 0 82.8%
Negative 10 4
SYBR Green I-EHP Positive 56 0 96.6%
Negative 2 4
3.8. Analyses of diagnostic specificity and diagnostic sensitivity of TaqMan-
based EHP qPCR, nested PCR and SYBR Green I qPCR-EHP
The diagnostic sensitivity, diagnostic specificity, and Kappa value
were compared among the detection results of 62 samples collected
from Zhoushan and Zhejiang Province by nested PCR, SYBR Green I
qPCR, and TaqMan qPCR. The results showed that compared with
nested PCR and SYBR Green I qPCR-EHP, the diagnostic specificity of
TaqMan qPCR was both 100%, the diagnostic sensitivity of nested PCR
and SYBR Green I qPCR-EHP was, 82.8% and 96.6%, respectively; the
Kappa value was 0.38 and 0.78, respectively. It was indicated that
TaqMan-based EHP qPCR had a higher detection conformity with SYBR
Green I-EHP, but a lower conformity with nested PCR (Table 4), which
might be due to the diagnostic sensitivity of TaqMan-based EHP qPCR,
which was close to but a little higher than SYBR Green I while that of
nested PCR was low.
3.9. The correlation between EHP loads in hepatopancreas and growth of P.
vannamei by TaqMan-based EHP qPCR detection
Body length and weight of each individual in P. vannamei samples
collected from four farms in Tianjin and Zhoushan, Zhejiang were
measured and EHP was detected with qPCR-EHP. The two batches of
samples from Farm 1 of Tianjin were collected before and after a ten-
tative medication to treat EHP, respectively. Negative correlation of
EHP loads with BMI was observed in both batches of samples, wherein
the correlation coefficients before and after the treatment were
R2 = 0.2792 and R2 = 0.6845, respectively (Fig. 3A). Mean of loga-
rithmic mean value of EHP loads per ng HpDNA in hepatopancreas of P.
vannamei before the treatment was 3.88 ± 0.58, and after the treat-
ment was 1.84 ± 1.50, which was 48.4 ± 39.4% of the value before
the treatment.
The correlation coefficient of the samples collected from Farm 2 of
Tianjin was 0.0053 (Fig. 3B), and that of the samples from Zhejiang
Province was 0.0053 (Fig. 3C), showing that there was almost no cor-
relation of EHP loads with BMI of P. vannamei.
4. Discussion
EHP was first found in P. monodon cultured in Thailand. At present,
it has been prevalent in Vietnam, China, Indonesia, Malaysia, Thailand,
and other countries. Due to determining the content of nucleic acid
accurately and specifically within a wide dynamic range, qPCR has
become the mainstream technology in detecting various DNA or RNA
pathogens in recent years. However, there was no TaqMan probe-based
qPCR method been published for EHP detection.
A TaqMan probe-based qPCR assay for EHP was established in this
study. It shows a good linearity in detecting standards of EHP SSU rDNA
ranging from 4 × 101 to 4 × 108 copies/µL using an established
method, and good intra-assay and inter-assay repeatability were ob-
served within the linearity range. Therefore, in detecting farm samples,
the positive detection rate of TaqMan-based EHP qPCR (93.5%) was
obviously higher than nested PCR (77.4%), while it was a little higher
than SYBR Green I qPCR-EHP (88.7%). Compared with nested PCR and
5
Journal of Invertebrate Pathology xxx (xxxx) xxx–xxx
Comparison with TaqMan-based EHP qPCR
Diagnostic sensitivity (DSe) Diagnostic specificity (DSp) Kappa value
100% 0.38
100% 0.78
SYBR Green I qPCR-EHP, the diagnostic specificity of TaqMan-based
EHP qPCR was 100%, while that of nested PCR and SYBR Green I qPCR-
EHP were 82.8% and 96.6%, respectively, indicating that TaqMan-
based EHP qPCR had a higher diagnostic sensitivity. The correlation of
the quantitative results of TaqMan-based EHP qPCR and SYBR Green I
qPCR-EHP was high and the value comparability was good. Although
SYBR Green I fluorochrome method is applied to the quantitative
analysis of DNA, the lack of specific fluorescent probe could cause in-
terference of primer dimers and nonspecific amplification products,
thereby, affecting the accuracy of results and even giving false posi-
tives. The method has rarely been used as a standard method for the
quantitative detection of pathogens in OIE aquatic animal diagnostic
standards. Therefore, the SYBR Green I qPCR assay was established in
our laboratory to detect EHP in former research (Liu et al., 2016);
however, the TaqMan qPCR assay was established on that basis in this
study. Through the comparison, the assay established in this study had
a higher sensitivity and specificity than SYBR Green I and nested PCR,
which could guarantee the sensitivity and accuracy of quantitative
determination of EHP.
The quantitative result showed that EHP loads had a negative cor-
relation with BMI in the samples collected from Farm 1 of Tianjin,
which indicated that EHP influenced the growth state of P. vannamei.
The same conclusion was reported by Liu et al. using the SYBR Green I
qPCR method in 2016. This study also demonstrated that EHP loads
appeared to obviously decrease after some treatment for EHP, and the
logarithmic mean value of EHP loads after the treatment dropped to
48.4 ± 39.4% of the value before the treatment, which indicated that
the qPCR assay established in this study could also be used in the
evaluation of EHP treatment. However, the correlation of EHP loads
and the BMI was not always negative. In the other samples collected
from Farm 2 of Tianjin and Zhejiang Province, EHP loads had no ob-
vious correlation with BMI, body length and weight, but these P. van-
namei with slow growth were all EHP-positive. Low correlation was also
observed in several samples between EHP loads and body length, when
using SYBR Green I qPCR in previous studies (Liu et al., 2016). The
above results indicated that EHP infection may affect the growth of P.
vannamei, but the EHP loads in the hepatopancreas might not always
have a correlation with the body length. It may be because of the dif-
ferent infection time of EHP in different individuals. The mechanism by
which EHP causes retarded growth in P. vannamei needs to be addressed
in future studies.
Acknowledgments
The authors wish to thank the farms in Tianjin and Zhejiang
Province in China for providing the P. vannamei samples. This study was
funded by the China Agriculture Research System (CARS-47), Project of
the Aoshan Sci. & Tech. Innovation Program of Qingdao National
Laboratory for Marine Science and Technology (Grant: 2015ASKJ02),
and the special foundation under the Construction Programme for
“Taishan Scholars” of Shandong Province.
Y.-M. Liu et al.
Fig. 3. A: Correlation of BMI and logarithmic EHP-copies per nanogram HpDNA of P. vannamei collected from a farm in Tianjin Province. B: Correlation of BMI and logarithmic EHP-
copies per nanogram HpDNA of P. vannamei collected from a farm in Tianjin Province. C: Correlation of BMI and logarithmic EHP-copies per nanogram HpDNA of P. vannamei collected
from a farm in Zhejiang Province.
Conflict of interest
All authors declare that there is no conflict of interest or financial
interest.
References
Amornrat, T., Jiraporn, S., Saisunee, C., Montagan, S., Niti, C., Chalor, L., Thinnarat, S.,
Timothy, W., Kallaya, S., 2013. The microsporidian Enterocytozoon hepatopenaei is not
the cause of white feces syndrome in whiteleg shrimp Penaeus (Litopenaeus) vannamei.
BMC Vet. Res. 9 (1) 139-139.
Liu, Z., Zhang, Q.L., Wan, X.Y., Ma, F., Huang, J., 2016. Development of real-time PCR
assay for detecting microsporidian enterocytozoon hepatopenaei and the application
in shrimp samples with different growth rates. Mar. Fish. Res. 37, 119–126.
OIE, 2015. Manual of Diagnostic Tests for Aquatic Animals. World Organisation of Animal
Health. < http://www.oie.int/international-standard-setting/aquatic-manual/
access-online/ > .
OIE, 2016. Manual of Diagnostic Tests for Aquatic Animals. World Organisation of Animal
Health. < http://www.oie.int/international-standard-setting/aquatic-manual/
access-online/ > .
Journal of Invertebrate Pathology xxx (xxxx) xxx–xxx
Suebsing, R., Prombun, P., Srisala, J., Kiatpathomchai, W., 2013. Loop-mediated iso-
thermal amplification combined with colorimetric nanogold for detection of the
microsporidian Enterocytozoon hepatopenaei in penaeid shrimp. J. Appl. Microbiol.
114, 1254–1263.
Tang, K.F., Pantoja, C.R., Redman, R.M., Han, J.E., Tran, L.H., Lightner, D.V., 2015.
Development of in situ hybridization and PCR assays for the detection of
Enterocytozoon hepatopenaei (EHP), a microsporidian parasite infecting penaeid
shrimp. J. Invertebr. Pathol. 130, 37–41.
Tangprasittipap, A., Srisala, J., Chouwdee, S., Somboon, M., Chuchird, N., Limsuwan, C.,
Srisuvan, T., Flegel, T.W., Sritunyalucksana, K., 2013. The microsporidian
Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg
shrimp Penaeus (Litopenaeus) vannamei. BMC Vet. Res. 9, 139–148.
Tourtip, S., Wongtripop, S., Stentiford, G.D., Bateman, K.S., Sriurairatana, S., Chavadej,
J., Sritunyalucksana, K., Withyachumnarnkul, B., 2009. Enterocytozoon hepatopenaei
sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp
Penaeus monodon (Decapoda: Penaeidae): fine structure and phylogenetic relation-
ships. J. Invert. Pathol. 102, 21–29.
Yue, Z.Q., Liu, H., Wang, W., Lei, Z.W., Liang, C.Z., Jiang, Y.L., 2006. Development of
real-time polymerase chain reaction assay with TaqMan probe for the quantitative
detection of infectious hypodermal and hematopoietic necrosis virus from shrimp. J.
AOAC Int. 89, 240–244.