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OPEN Detection of non‑pathogenic

and pathogenic populations
of Vibrio parahaemolyticus
in various samples
by the conventional, quantitative
and droplet digital PCRs
Sinisa Vidovic 1*, Roland Taylor 1, Duncan Hedderley 2, Graham C. Fletcher 1 & Nicola Wei 3

In this study, three generations of polymerase chain reaction (PCR) assays: (i) conventional PCR, (ii)
qPCR and (iii) droplet digital PCR (ddPCR), were systematically tested for their abilities to detect
non-pathogenic and pathogenic populations of Vibrio parahaemolyticus. The limit of detection (LOD)
for the ddPCR was 1.1 pg/µL of purified DNA, followed by the qPCR (5.6 pg/µL) and the conventional
PCR (8.8 pg/µL). Regarding the LOD for V. parahaemolyticus cells, the ddPCR assay was able to
detect 29 cells, followed by the conventional PCR assay (58 cells) and the qPCR assay (115 cells).
Regarding the sensitivities to detect this pathogen from PCR inhibition prone samples (naturally
contaminated mussels), the ddPCR assay significantly outperformed the conventional PCR and qPCR.
The ddPCR assay was able to consistently detect non-pathogenic and pathogenic populations of
V. parahaemolyticus from naturally contaminated mussels, indicating its tolerance to various PCR
inhibitors. This study also revealed the significant difference between conventional PCR and qPCR.
The conventional PCR assay showed significantly greater sensitivity than that of the qPCR assay in
detecting V. parahaemolyticus in crude samples, whereas the qPCR assay showed better sensitivity in
detecting the presence of V. parahaemolyticus in purified DNA samples.

Keywords Vibrio parahaemolyticus, Emerging pathogen, Vibriosis, Diagnostics, Seafood safety

Vibrio parahaemolyticus, a Gram-negative halophilic bacterium, is ubiquitously present in estuarine marine

and coastal environments. Although many strains of V. parahaemolyticus can be harmless to humans, cer-
tain pathogenic strains of this bacterial species can cause gastroenteritis, septicaemia, and wound ­infections1.
Indeed, V. parahaemolyticus is the leading cause of gastrointestinal infections associated with the consumption
of seafood ­worldwide2–4. Vibriosis caused by V. parahaemolyticus is mainly associated with symptoms such as
fever, headache, vomiting, abdominal cramps and diarrhoea. The gastroenteritis-type infection is self-limiting5.
V. parahaemolyticus outbreaks are mainly associated with the consumption of molluscan shellfish, a concern
for public health and the seafood industry in many parts of the w ­ orld6–8. Not only have the frequencies of these
outbreaks increased in the last two ­decades , but also outbreaks associated with V. parahaemolyticus have been
9

recorded in geographic regions that previously never experienced such ­outbreaks10. New Zealand has experienced
outbreaks of vibriosis in winter ­months11, when concentrations of V. parahaemolyticus in shellfish are invari-
ably ­low7. Therefore, the employment of sensitive and reliable diagnostic methods for the detection of the total
population of V. parahaemolyticus and its more pathogenic sub-population12–16 plays an important role in the
prevention of this food-borne infectious disease.
Two major virulence factors, thermostable direct hemolysin (TDH) and thermostable-related hemolysin
(TRH), are commonly associated with the clinical ­isolates17. Specifically, TDH, which produces ß-type haemolysis

1
The New Zealand Institute for Plant and Food Research Limited, 120 Mount Albert Road, Sandringham,
1025 Auckland, New Zealand. 2The New Zealand Institute for Plant and Food Research Limited, Palmerston North,
New Zealand. 3McGill University, Montreal, Canada. *email: sinisa.vidovic@plantandfood.co.nz

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on Wagatsuma agar, is commonly associated with the population of clinical V. parahaemolyticus isolates, but it
is less commonly present among environmental i­ solates18,19, confirming its importance in the pathogenicity of
V. parahaemolyticus. In addition to these two haemolysins, V. parahaemolyticus possess two type III secretion
systems, T3SS1 and T3SS2, responsible for invasion of the host cells and survival of the pathogen within the host
­cell20. In general, T3SS1 is commonly present in V. parahaemolyticus isolates and plays a role in ­autophagy21 and
­cytotoxicity22. In contrast, T3SS2 is rarely found among populations of V. parahaemolyticus, and it is suspected
that this secretion system plays a role in e­ nterotoxicity22.
Reliable diagnostic data are crucial for development of effective measures to prevent outbreaks associated
with this organism. Therefore, we carried out a study to validate three molecular assays for the detection of V.
parahaemolyticus in different sample matrix. These diagnostic assays are based on three different PCR platforms,
including conventional PCR, quantitative PCR (qPCR), and droplet digital PCR (ddPCR). The PCR-based assays
were simultaneously tested for the detection of species and pathogen-specific markers of V. parahaemolyticus.
The assays were tested using three laboratory-based approaches: (i) genomic DNA, (ii) viable cells of V. para-
haemolyticus, and (iii) spiked muscle tissue. Besides the laboratory-based approaches, all PCR assays were tested
against 480 freshly harvested mussels for the presence of V. parahaemolyticus.

Material and methods

Strains of V. parahaemolyticus
Two strains of V. parahaemolyticus of clinical origin were used for the laboratory-based studies. The first strain,
19ER2200 (tdh and trh positive), was obtained from the Institute of Environmental Science and Research (ESR),
New Zealand. This strain was the causative agent of the first V. parahaemolyticus outbreak in New Zealand, in
June 2019. The second strain, F11-3A (tdh and trh positive) isolated in Washington state, USA, was associated
with a 1997 US outbreak. Both strains belong to the pandemic sequence type (ST)36. Pseudomonas fluorescens
strain cc848406E was used as a negative control. The isolates were stored at – 80 °C in Luria–Bertani (LB) broth
(Difco) with 10% glycerol. For each laboratory-based experiment, fresh cultures derived from the frozen stocks
were used.

Diagnostic PCR assays

Three multiplex PCR platforms, a conventional multiplex ST36 ­PCR14, a multiplex ­qPCR15, and a multiplex
­ddPCR16, were tested for their sensitivities to detect V. parahaemolyticus. Oligonucleotide primers and probes
used in this study are presented in Table 1.
Conventional and ddPCR were carried out using the T100 and C1000 ­Touch™ thermal cyclers (Bio-Rad,
USA), respectively while the qPCR assay was carried out using the 7500 Fast Real-Time PCR System (Applied
Biosystems). Droplets for ddPCR were generated with the automated droplet generator (Bio-Rad, USA). The PCR
­ escribed14,15.
reactions were carried out in a 20 µL reaction mixture using 2 µL of PCR template, as previously d
Only the ddPCR assay was slightly modified. Briefly, the PCR reaction mixture (20 µL) contained 2 × ddPCR
Supermix for Probes (no dUTP) (Bio-Rad, USA), 990 nM of all three pair of primers, and 250 nM of ureR (FAM
1), 125 nM of tlh (FAM 2), as well as 500 nM of tdh (HEX) probes. The samples were treated for 10 min at 95 °C,
following 39 cycles at 94 °C for 30 s and 56 °C for 60 s and one cycle at 98° C for 10 min. The digital droplets
were detected by the QX ­200™ Droplet Reader (Bio-Rad, USA) and analysed by QX Manager software, standard
edition, version 2.0 (Bio-Rad, USA). Each laboratory-based experiment was carried out on three different occa-
sions (biological replications) using two technical replications for each sample.

Gene Type of PCR assay Forward primer sequence (5′–3′) Reverse primer sequence (5′–3′) Reference
tlh AGA​ACT​TCA​TCT​TGA​TGA​CAC​TGC​ GCT​ACT​TTC​TAG​CAT​TTT​CTC​TGC​ 14

tdh GTA​AAG​GTC​TCT​GAC​TTT​TGGAC​ TGG​AAT​AGA​ACC​TTC​ATC​TTC​ACC​ 31

trh ST36 PCR CAT​AAC​AAA​CAT​ATG​CCC​ATT​TCC​G TTG​GCT​TCG​ATA​TTT​TCA​GTA​TCT​ 31

prp CGG​CTT​GAG​TTT​TCG​TCA​TT CCA​CAC​CTG​CTG​GTT​ATT​TAG​TTC​ 14

flp TGG​TTG​TGT​TTA​GAG​CAG​GG TGT​TGG​TAA​TAC​GAT​AAG​AAT​GAG​A 14

tlh ACT​CAA​CAC​AAG​AAG​AGA​T CGA​CAA​ GAT​GAG​CGG​TTG​ATG​TCC​AA 15

tlh probe CGC​TCG​CGT​TCA​CGAAA CCGT​ 15

trh TTG​CTT​TCA​GTT​TGC​TAT​TGGCT​ TGT​TTA​CCG​TCA​TAT​AGG​CGCTT​ 15

qPCR
trh probe AGA​AAT​ACA​ACA​ATC​AAA​ACTGA​ 15

tdh TCC​CTT​TTC​CTG​CCCCC​ CGC​TGC​CAT​TGT​ATA​GTC​TTT​ATC​ 15

tdh probe TGA​CAT​CCT​ACA​TGA​CTG​TG 15

tlh GAA​CGC​AGA​CAT​TACG​ ACC​ACT​TTG​TTG​ATT​TGA​ 16

tlh probe CAT​TGC​TGC​GTC​GTT​GCT​CC 16

tdh GGT​CAG​GAA​GTT​CGT​ ACG​GCA​TAG​GTG​AGTA​ 16

ddPCR
tdh probe CCG​CCA​CGA​CAG​TTA​CGA​ 16

ureR GCA​CTC​TAA​CAC​CCAA​ AGC​TGA​TAC​ATC​GGTT​ 16

ureR probe CTA​GGC​GAG​CAA​AAG​CAC​TCT​ 16

Table 1.  Oligonucleotide primers used for the PCR assays.

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Extraction and preparation of genomic DNA

Vibrio parahaemolyticus strains were cultured from frozen stocks on Tryptone Soy Agar (TSA) plates (Difco) sup-
plemented with 2% NaCl followed by overnight incubation at 35 °C. Three single colonies were picked by a sterile
cotton swab and resuspended in 1 mL of 0.9% saline. After centrifugation at 10,000×g for 5 min, the supernatant
was removed and genomic DNA was extracted using a Qiagen DNeasy Tissue kit (Qiagen Inc., Valencia, CA)
according to the manufacturer’s instructions. After this, the genomic DNA from each ST36 strain, F11-3A and
19ER2200, was equally combined (v/v), followed by serial dilutions using fivefold dilution steps. The first three
concentrations of DNA were determined with a N ­ anoDrop™ microvolume spectrophotometer, whereas the rest
of the dilutions were calculated since these DNA concentrations went beyond the sensitivity limit of the Nan-
oDrop instrument. The DNA concentration of each PCR template used in this experiment is indicated in Table 2.

Preparation of viable V. parahaemolyticus cell PCR templates

The inoculum was prepared using overnight cultures of each ST36 strain grown in Alkaline Peptone Water
(APW)23, broth at 35 °C with constant shaking at 180 rpm. Seed cultures were diluted 1/100 in 100 mL of freshly
prepared APW broth and grown to an optical density at 600 nm of 0.4. After this, the cultures were centrifuged
and washed three times then adjusted to 0.5 McFarland standard, which is comparable to a bacterial suspension
of ~ 1.5 × ­108 colony-forming units (CFU) per 1 mL. After normalization of cultures, equal volumes of each ST36
strain culture were added and mixed to produce a final inoculum. This final inoculum was serially two-fold
diluted, resulting in ten different cell dilutions. Cell numbers of inocula were determined by serial dilutions and
spread plating on TSA supplemented with 2% of NaCl. Inocula cell numbers per mL and PCR reactions of each
PCR template used in this experiment are provided in Table 3.

Spiking of mussel tissue

The inocula were prepared as described above. In total, seven different inocula dilutions were prepared using
tenfold dilution steps. Cell numbers of inocula were determined as above and dilutions as well as cell numbers of
inocula used for each biological replication are shown in Table 4. Once the inocula were prepared, 100 μL of V.
parahaemolyticus suspension was added to 1 mL of homogenised raw mussel tissue (Table 4) followed by a brief
mixing and incubation at room temperature for 1 h. After this brief incubation, 9 mL of APW broth was added

DNA concentrations
Sample dilutions First biological replication Second biological replication Third biological replication
1/5 dilution 67.2 ng/µL 87.3 ng/µL 29.3 ng/µL
1/25 dilution 13.5 ng/µL 17.1 ng/µL 7 ng/µL
1/125 dilution 3.5 ng/µL 4.3 ng/µL 1.1 ng/µL
1/625 dilution 700 pg/µL 860 pg/µL 220 pg/µL
1/3125 dilution 140 pg/µL 172 pg/µL 44 pg/µL
1/15,625 dilution 28 pg/µL 34.4 pg/µL 8.8 pg/µL
1/78,125 dilution 5.6 pg/µL 6.88 pg/µL 1.8 pg/µL
1/390,625 dilution 1.1 pg/µL 1.4 pg/µL 352 fg/µL
1/1,953,125 dilution 224 fg/µL 275 fg/µL 70.4 fg/µL
1/9,765,625 dilution 44.8 fg/µL 55 fg/µL 14 fg/µL

Table 2.  Concentration of Vibrio parahaemolyticus DNA samples used for PCR templates.

First biological replication Second biological replication Third biological replication

Cells per PCR
Sample dilutions Cells per mL Cells per PCR reaction Cells per mL Cells per PCR reaction Cells per mL reaction
1/2 230,000 460 730,000 1460 400,000 800
1/4 115,000 230 365,000 730 200,000 400
1/8 57,500 115 182,500 365 100,000 200
1/16 28,750 57.5 91,250 182 50,000 100
1/32 14,375 29 45,625 91 25,000 50
1/64 7187 14 22,812 46 12,500 25
1/128 3594 7 11,406 23 6250 12.5
1/256 1797 3.5 5703 11 3125 6
1/512 898 1.8 2851 5.7 1562 3
1/1024 449 0.9 1425 2.8 781 1.5

Table 3.  Numbers of Vibrio parahaemolyticus cells for each inoculum and PCR template.

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First biological replication Second biological replication Third biological replication

a
Concentration of DNA a
Concentration of DNA a
Concentration of DNA
Fold dilutions Inoculum/cells per mL (ng/µL) Inoculum/cells per mL (ng/µL) Inoculum/cells per mL (ng/µL)
10−4 15,600 122.6 6600 60.7 6300 191.8
10−6 156 67.9 66 126.3 63 86.2
10−7 16 80.1 6.6 116 6.3 85.8
10−8 1.6 49.1 0.66 36.8 0.63 22.8
10−9 0.16 27.6 0.066 41.9 0.063 27.4
10−10 0.016 35.3 0.0066 18.7 0.0063 29
10−11 0.0016 18 0.00066 16 0.00063 36.8

Table 4.  Cell numbers used for Vibrio parahaemolyticus inoculation of mussel tissues. a Concentrations of
DNA extracted from 1 mL of spiked and incubated mussel samples.

to each spiked mussel sample followed by overnight incubation at 35 °C. After overnight incubation, 1 mL of
suspension was taken and centrifuged at 10,000×g for 3 min. The supernatant was removed, and genomic DNA
was extracted using a Qiagen DNeasy Tissue kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s
instructions. The concentration of DNA extracted from each spiked mussel sample is shown in Table 4.

Collection of mussels
Greenshell™ mussels (Perna canaliculus) were collected at an aquaculture farm in the Marlborough Sounds, New
Zealand, over three summer months (January 2022, February and January 2023) and also over three winter
months in 2022 (June, July and August). In total, 480 mussels were collected. Each month, 80 mussels were
harvested and placed inside a polystyrene box with a coolant pad and delivered to The New Zealand Institute
for Plant and Food Research Limited, Auckland, within 24 h.

Processing of mussels, incubation, and DNA extraction

Immediately upon arrival, the exterior shell surface of each mussel was wiped using Tork Xpress paper hand
towels then the weight and the total length of each mussel was recorded. The mussels were aseptically shucked
and tissues of 13 mussels, including their intervalvular water, were homogenised using a pre-weighed sterile
Waring blender cup, followed by seven decimal dilutions of the homogenised tissue in Phosphate Buffered
Saline (PBS, pH 7.4). Each dilution was transferred into triplicate tubes of APW starting with 1 g of undiluted
homogenate into 9 mL of APW. Suspensions of mussel tissues and APW were incubated for 24 h at 35 °C for
enrichment. After the first 24 h, 1 mL of the culture from the first three dilutions of each series was subsampled
into fresh 10 mL APW tubes and incubated for a further 24 h at 35 °C24. This was done to limit of the amount of
mussel meat and other potential PCR reaction inhibitors in the least-diluted tubes. After incubation, 1 mL was
taken from each turbid enrichment, followed by DNA extraction using the heat boiling m ­ ethod25. Briefly, 1 mL
of enrichment suspension was centrifuged at 15,000×g for 10 min. The supernatant was removed, and the pellet
was resuspended in 1 mL of molecular grade water followed by a second centrifugation at 15,000×g for 10 min.
The resulting pellet was resuspended in 50 µL of molecular grade water, followed by boiling at 100 °C for 10 min.
After the boiling, the samples were cooled on ice and stored at − 20 °C for further use.

Statistical analysis
For the ddPCR assay, for each of the three types of samples (genomic DNA, V. parahaemolyticus cells, and mussel-
tissue spiked with V. parahaemolyticus), a dilution level was identified which appeared to give signals just above
the signal from the blank wells. The signals of three genes (ureR, tlh and tdh) from positive but diluted samples
were compared with those from blank wells graphically and using Receiver Operating Characteristic (ROC)
curves calculated using the R package p ­ ROC26. Limits of detection were calculated as the upper 95% confidence
limit for signals from the blank cells, using a t-distribution.

Results
Sensitivity of the diagnostic assays to detect extracted and purified genomic DNA
The sensitivities of the PCR platforms were determined using three biological and two technical replications
with five-fold serially diluted DNA samples (Table 2). The results associated with biological replications where
particular PCR assay achieved the lowest detection limit are presented. The multiplex ST36 PCR assay could
detect all five genes, indicating the presence of non-pathogenic (tlh), pathogenic (tdh, trh) and ST36 (prp, flp)
specific gene markers, in a sample containing 8.8 picograms (pg) of total DNA (Fig. 1A). This DNA sample was
1/15625 diluted compared with its original sample concentration and presented the lowest possible concentra-
tion of DNA that could be detected by the multiplex ST36 PCR assay (Fig. 1A). Using the same set of DNA
samples, it was found that ddPCR and qPCR assays could detect the presence of virulence gene (tdh) in a sample
containing 1.1 pg of DNA (1/390625 dilution) (Fig. 1B, C). However, the qPCR assay could not detect the pres-
ence of a non-pathogenic gene marker (tlh) in that sample (Fig. S1), whereas the ddPCR assay detected both
the tlh gene (species-specific) and the tdh and ureR genes (pathogenic markers) (Fig. 1B), further indicating the
presence of this non-pathogenic gene marker. To obtain reliable data indicating a limit of detection (LOD) for

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Figure 1.  Sensitivity limits of PCR Vibrio parahaemolyticus assays as determined with serially diluted DNA.
(A) Gel image shows the sensitivity limit of a multiplex ST36 PCR assay. Note, each DNA dilution was carried
out in two technical replications. (B) ddPCR graph shows the presence and distribution of positive tlh (blue),
ureR (green), tdh (red), tdh and ureR (orange) as well as negative (grey) droplets. The PCR reaction was carried
out with 1.1 pg of genomic DNA. (C) Sensitivity of the qPCR assay using the last five dilution series (28 pg/µL,
5.6 pg/µL, 1.1 pg/µL, 224 fg/µL and 44.8 fg/µL) and targeting the tdh virulence marker. The Ct cut-off value (30)
is indicated by the arrow. The qPCR was done in duplicate for each sample tested.

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the ddPCR, the most sensitive assay, an additional six (third biological replication) and seven (first and second
biological replications) technical replications, including negative controls, were carried out using a set of samples
containing 1.1 pg of DNA (first biological replication), 1.4 pg of DNA (second biological replication) and 352 fg
of DNA (third biological replication). This analysis showed that the copies of all three gene markers (tlh, tdh
and ureR) for the biological replications one and two were significantly above those of negative controls (Fig. S2
and Table S1). In contrast, for the copies of the three genes associated with the biological replication three were
not distinguishable from those of the negative controls (Fig. S2 and Table S1). In other words, the ddPCR could
detect all three genomic markers in a sample containing 1.1 pg of DNA, whereas the detection signals were lost
in a sample containing 0.3 pg (352 fg) of DNA.
In summary, the ddPCR and qPCR assays showed a similar level of sensitivity in the detecting genomic DNA
of V. parahaemolyticus, whereas the conventional ST36 PCR assay exhibited a lower sensitivity.

Detection of viable V. parahaemolyticus cells

Using viable cells of V. parahaemolyticus as a PCR template (a colony-PCR approach), the qPCR assay was able
to detect the presence of non-pathogenic (tlh) and pathogenic (tdh) markers in a sample containing 115 cells
(Fig. 2A). The conventional ST36 PCR assay could detect the presence of this food-borne pathogen in a sample
containing 57 cells (Fig. 2B), a better level of sensitivity than the qPCR assay. Testing the sensitivity of the ddPCR
assay on the same set of samples, it was found that this PCR assay could detect the presence of pathogenic (tdh
and ureR) and non-pathogenic (tlh) markers in a sample containing 29 cells (Fig. 2C). To increase the robustness
of the data for the ddPCR, an additional seven (third biological replication) and eight (first and second biological
replications) technical replications of each biological replication were tested, including samples that contained 29
cells (first biological replication), 91 cells (second biological replication) and 50 cells (third biological replication).
This additional analysis showed that the ddPCR assay could detect 0.7 copies of ureR, 1.09 copies of tlh and 0.93
copies of tdh per 1 µL of the sample that contained 29 cells in total, which was significantly above the detection
in the negative control samples (Fig. S3 and Table S2). The colony-PCR approach demonstrated that the ddPCR
was the most sensitive PCR platform in detecting viable cells of V. parahaemolyticus.

Detection of V. parahaemolyticus in spiked mussel samples

After conducting the tests using three biological replications of spiked mussel tissue, the conventional ST36 PCR
assay detected all five genetic markers (tdh, tlh, trh, prp and flp) in the samples inoculated with 1­ 0−7 inoculum
(Table 4) or 16 cells (Fig. 3A). After this, no diagnostic bands could be detected in the samples inoculated with
more diluted inocula (Fig. 3A), indicating that the ­10−7 inoculum was the LOD for the conventional ST36 PCR
assay. In the case of the qPCR assay, a strong detection signal (10–15 Ct) was observed in the samples inoculated
with the first three inocula (­ 10−4, ­10−6 and 1­ 0−7), whereas in the rest of the samples, inoculated with more diluted
inocula ­(10−8, ­10−9, ­10−10 and ­10−11), was observed a weak positive diagnostic signal (25–30 Ct) (Fig. 3B). Using
the identical set of samples, the ddPCR assay also showed the presence of V. parahaemolyticus in all samples.
The diagnostic signal for the samples inoculated with the most diluted inoculum ­(10–11) for all three biological
replications was in the ranges of 2.71 to 5.4 copies per µL for the ureR gene, 4.37 to 10.3 copies per µL for the tlh
gene, and 2.91 to 6.31 copies for the tdh gene (Fig. 3C). Conducting an additional ddPCR analysis that included
eight technical replications for each biological replication of the samples inoculated with the most diluted inocu-
lum, showed that the mean value ranged for the ureR gene from 1.52 to 5.82 copies, for the tlh gene between
3.18 and 12.25 copies, and for the tdh gene the mean value ranged from 1.78 to 6.40 copies per 1 µL (Fig. S4 and
Table S3). These mean values were significantly above those of the negative control values (from 0.10 to 0.13
copies), clearly indicating the presence of V. parahaemolyticus. The experiment with the spiked mussel tissue
showed that the conventional ST36 PCR assay was the least sensitive, whereas the qPCR and ddPCR assays
showed greater sensitivities.

Ability of the diagnostic assays to detect V. parahaemolyticus in naturally contaminated

mussels
The naturally contaminated mussels were harvested during two distinct temperature periods in the Southern
Hemisphere, in winter months (June–July, and August) and summer months (January 2022, February, and Janu-
ary 2023). In general, during the winter months, the prevalence of V. parahaemolyticus in mussels was signifi-
cantly lower than in the summer months (Fig. 4A, B). Bacterial DNA from all mussel samples was extracted by
the boiling method. For the mussels harvested in June, the ddPCR assay detected 37.5% (n = 27) positive samples
for V. parahaemolyticus (tlh), while the conventional ST36 PCR assay detected 19.4% (n = 14) and qPCR showed
2.7% (n = 2) of samples positive for V. parahaemolyticus (Fig. 4A). Similarly, the ddPCR assay detected 37.5%
(n = 27) of samples being positive for the virulence factor, tdh, whereas the conventional ST36 PCR indicated 18%
(n = 13) and the qPCR showed 1.3% (n = 1) of samples being positive for the same virulence marker (Fig. 4A).
For the samples harvested in July, the ddPCR assay detected 26.9% (n = 21) of samples being positive for the
presence of V. parahaemolyticus (tlh) and its virulence marker (tdh), while the conventional ST36 PCR showed
15.4% (n = 12) of samples positive for both tlh and tdh (Fig. 4A). The qPCR assay detected neither tlh nor tdh in
the same set of samples. For the last winter month, August, the ddPCR identified 16.2% (n = 13) tlh-positive and
17% (n = 14) tdh-positive samples, whereas the conventional ST36 PCR showed 10% (n = 8) positive samples for
tlh and 2.5% (n = 2) positive for tdh (Fig. 4A). The qPCR could detect neither tlh nor tdh in mussels harvested
during August (Fig. 4A). For the first time, testing the mussels harvested in January 2022, the conventional ST36
PCR assay showed a greater sensitivity, detecting 89.7% (n = 61) of samples positive for V. parahaemolyticus
compared with 77.9% (n = 53) positive samples identified by the ddPCR assay (Fig. 4B). The conventional ST36
PCR assay detected 14.7% (n = 10) of samples with the tdh virulence marker, whereas the ddPCR assay showed

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Figure 2.  Sensitivity limits of PCR assays as determined with viable Vibrio parahaemolyticus cells (colony-PCR
approach). (A) Amplification plot showing the limit of detection for the qPCR assay. The graph shows the results
for four different samples each containing 460 cells, 230 cells, 115 cells and 57.5 cells, respectively. The red arrow
indicates the cut-off value for this PCR assay. (B) Gel image portraying the limit of detection for the multiplex
ST36 PCR assay. (C) ddPCR graph showing the presence and distribution of the positive tlh (dark blue), ureR
(green), tdh (red), tlh and ureR (light blue), tdh and ureR (purple), tlh, tdh and ureR (orange) and negative (grey)
droplets. The template contained 29 cells of V. parahaemolyticus.

76.4% (n = 52) of samples being positive for the tdh gene (Fig. 4B). The qPCR assay detected 4.4% (n = 3) of
samples as positive for V. parahaemolyticus and no samples positive for the tdh virulence factor (Fig. 4B). For
the mussels harvested in February, the ddPCR assay detected 77.1% (n = 54) of samples positive for the presence
of V. parahaemolyticus and 48.5% (n = 34) of samples positive for the tdh gene (Fig. 4B). At the same time, the

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Figure 3.  Sensitivity limits of PCR assays with mussel tissues spiked with Vibrio parahaemolyticus. (A) Gel
image portraying the limit of detection for the multiplex ST36 PCR assay. (B) Amplification graph showing
the limit of detection for the qPCR assay. The graph shows the results for all samples, grouping them into
two groups: (i) strong positive (first three dilution series), and (ii) weak positive (the remaining four dilution
series). The red arrow indicates the cut-off value for this PCR assay. (C) ddPCR graph showing the presence and
distribution of the positive tlh (dark blue), ureR (green), tdh (red), tdh and tlh (purple) and tlh, tdh and ureR
(orange) and negative (grey) droplets.

conventional ST36 assay detected 58.6% (n = 41) of samples positive for V. parahaemolyticus and 24.3% (n = 17)
of samples positive for the tdh virulence factor (Fig. 4B). The qPCR assay identified 30% (n = 21) samples posi-
tive for V. parahaemolyticus and did not detect any presence of the tdh gene (Fig. 4B). For the mussels harvested
during January 2023, the ddPCR assay detected 88.3% (n = 76) of samples as positive for V. parahaemolyticus,

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Figure 4.  Sensitivities of conventional ST36 PCR, qPCR and ddPCR in the detection of Vibrio
parahaemolyticus from naturally contaminated mussels. (A) In winter months, mussels were harvested in
seawater that ranged from 11.8 °C (July) and 12.2 °C (August) to 14.6 °C (June). (B) During summer months,
mussels were harvested in seawater that ranged from 18.7 °C (January) to 19.5 °C (February).

followed by the conventional ST36 PCR assay, 71% (n = 61) and the qPCR assay, 51.1% (n = 44) (Fig. 4B). A similar
trend was observed in the detection of the virulence factor, tdh, with the ddPCR assay being the most sensitive,
resulting in 90.7% (n = 78) of positive samples, followed by the conventional ST36 PCR with 50% (n = 43), and
the qPCR assay with 15.1% (n = 13) positive samples, respectively (Fig. 4B).

Discussion
To reveal the strengths and weaknesses of different diagnostic methods employed for the detection of V. para-
haemolyticus, it is important to evaluate their sensitivities using various testing approaches. In this study, the
sensitivities of three PCR assays, based on three platforms, (i) conventional PCR, (ii) qPCR and (iii) ddPCR,
were determined against sets of samples containing purified genomic DNA, viable cells, spiked mussel tissue,
and naturally contaminated mussels. Also, two sample preparation methods for DNA extraction were used, the
Qiagen DNeasy Tissue kit and the heat boiling method.
Using purified genomic DNA derived from V. parahaemolyticus and testing the LOD of the employed PCR
assays, it was found that the ddPCR exhibited the greatest LOD, detecting the presence of V. parahaemolyticus
(tlh) and its virulence markers (tdh and ureR) in a sample containing 1.1 pg/µL of highly purified DNA. The
qPCR assay was capable of detecting V. parahaemolyticus in the same sample but failed to detect the targeted
pathogenic marker (tdh). Zhu and c­ olleagues27, using the real-time recombinase polymerase amplification PCR
approach for the detection of the tlh gene, reported 0.4 pg/µL of DNA as the LOD for this assay. Another group
of authors, employing a multiplex qPCR with TaqMan fluorescent probes, reported that 200 pg of purified
genomic DNA was the LOD for this PCR ­assay28. Note that the real-time recombinase polymerase amplification
assay targeted only a single genetic marker (tlh), whereas the qPCR with TaqMan fluorescent probes and the
PCR assays used in this study targeted multiple genetic markers simultaneously. It has been reported that the
use of the multiplex PCR approach causes competition between the amplifications of multiple genes, resulting in

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significant decreases of LOD for the employed multiplex PCR ­assays29. If one gene is significantly more abundant
compared to other genes in a multiplex reaction, this may lead to reaching its plateau phases much earlier. This
usually results in the limitation of nucleotides and other reagents for the remaining genes, causing their limited
amplification. The phenomenon of the competition between amplifications of multiple genes in the multiplex
PCR format may explain why the qPCR assay employed in our study was able to detect the tdh gene but could
not detect the presence of the tlh gene in a sample that contained the same number of the tdh and tlh gene copies.
The colony-PCR approach revealed that the ddPCR exhibited the greatest LOD, compared with those of the
conventional PCR and qPCR assays. Interestingly, the conventional PCR assay showed greater sensitivity than
the qPCR assay in detecting the viable V. parahaemolyticus cells, whereas the qPCR assay was more sensitive
than the conventional PCR in the detection of V. parahaemolyticus via the purified genomic DNA. These results
indicate that the type of genomic template can play a role in the overall sensitivity of different PCR assays in the
detection of V. parahaemolyticus. Indeed, Jones et al. (2012) observed a significant difference in the sensitiv-
ity of the real-time PCR a­ ssay29 in detecting V. parahaemolyticus when it was run using the B ­ AX® System lysis
(p = 0.0004) for the PCR template preparation versus the heat boiling method (p = 0.0347).
Testing the spiked mussel tissue samples, great difference was observed between the conventional PCR assay
on one hand and the ddPCR and qPCR assays on the other hand. While the conventional PCR assay was able to
detect the presence of V. parahaemolyticus and its pathogenic markers only in the mussel samples inoculated with
the first three inocula, the ddPCR and qPCR identified this organism together with the tdh gene in the samples
inoculated with all seven inocula. The literature commonly reports that food matrices, especially shellfish, contain
a wide range of potent PCR inhibitors that can potentially lead to the generation of false-negative ­results30–32. The
presence of PCR inhibitors is unlikely to have affected the conventional PCR assay as the spiked mussel tissue
samples underwent the enrichment step followed by the DNA extraction and purification, further ensuring a
high quality of PCR templates for this experimental approach. The most likely explanation for the weak sensitiv-
ity of the conventional PCR assay compared with that of the qPCR assay lies in the fact that the qPCR shows a
greater sensitivity with purified DNA, as already shown in the experiment with serially diluted genomic DNA.
Finally, the sensitivities of the PCR assays were determined against a large number of naturally contaminated
mussels that were harvested during the winter and summer months in a region known to produce low concentra-
tions of V. parahaemolyticus in mussels. A rationale for this experimental approach was to test the PCR assays
against mussels that were naturally contaminated with low (winter) and higher (summer) numbers of V. para-
haemolyticus cells. Overall, the ddPCR assay showed superiority over the other two PCR platforms in detecting
not only the non-pathogenic marker of V. parahaemolyticus but also the pathogenic markers over the winter
and summer months. Only once (testing mussels that were harvested in January 2022) was the conventional
PCR assay capable of exhibiting a slightly greater sensitivity than the ddPCR assay in detecting the presence of
V. parahaemolyticus (tlh). The initial testing of the mussels, harvested during the summer months, suffered an
inability of the ddPCR assay to generate separate clusters of positive droplets, rather generating a single cluster
positioned at the central point of the graph. This failure was caused by the high abundance of genetic material,
causing mispriming and/or loss of amplification. It has been reported that a high abundance of PCR templates
can lead to amplification failure or mispriming, resulting in overall PCR reaction ­failure33. Indeed, a dilution of
these samples led to the generation of distinct clusters with positive droplets, allowing absolute quantification
of the targeted gene loci. Regarding the other two PCRs platforms, the conventional PCR assay significantly
outperformed the qPCR assay in detecting the total and pathogenic populations of V. parahaemolyticus in natu-
rally contaminated mussels over the entire course of the study. It is important to mention that the detection of
V. parahaemolyticus from the naturally contaminated mussels was carried out using the heat boiling method.
This DNA extraction method does not purify genomic material and therefore a wide range of PCR inhibitors
can be present in these samples. From the literature is known that ddPCR exhibits high resilience to various PCR
inhibitors derived from plants, soil and wastewater compared to other PCR ­platforms34. The greater sensitivity
of the ddPCR in detecting V. parahaemolyticus from PCR inhibition prone samples can be explained by the high
resilience of ddPCR to various PCR inhibitors.
In summary, the sensitivities of three PCR assays, based on three PCR platforms, were systematically com-
pared using laboratory and environmentally based approaches. The ddPCR assay demonstrated consistently
great sensitivity in detecting V. parahaemolyticus in various types of samples, including PCR inhibition prone
samples. In contrast to the ddPCR assay, the conventional PCR and qPCR assays showed significant fluctuations
in their sensitivities, depending on the type of tested samples. In general, the conventional PCR assay showed
significantly greater sensitivity than that of the qPCR assay in detecting V. parahaemolyticus in crude samples
(naturally contaminated mussels and viable cells), whereas the qPCR assay showed a better sensitivity than the
conventional PCR in detecting the presence of V. parahaemolyticus in purified DNA samples (purified genomic
DNA and spiked mussels).

Data availability
Requests for access to the data for research purposes can be sent to Sinisa.Vidovic@plantandfood.co.nz.

Received: 8 January 2024; Accepted: 15 February 2024

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Acknowledgements
The authors are grateful for technical support from Daniela Vidovic and Sol Green. We also thank Zhiwei Luo
for his assistance. This work was funded by the New Zealand Ministry of Business, Innovation and Employment
through the Cawthron Institute’s Seafood Safety programme, contract CAWX1801.

Author contributions
Validation, Visualization, Writing—Original draft. R.T. Investigation, Formal analysis, Validation, Review and
Editing. D.H. Statistical analysis, Review and Editing. G.C.F. Conceptualization, Review and Editing. N.W. Inves-
tigation, Formal Analysis, Validation, Review and Editing.

Competing interests
The authors declare no competing interests.

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