Evaluation of Method of Visual Gene Chip Detection for Four

Pathogenic Bacteria in Aquatic Products

Lin Lu

National Institute of Metrology, China, Beijing 100029, China

Keywords: Aquatic Products, Pathogenic Bacteria, Visual Detection, Gene Chip, Evaluation.

Abstract: In this manuscript, the author developed a gene chip detection method that combined tyramine signal

amplification technology and nanogold-labeled silver staining technology. Four pathogenic bacteria (Listeria

monocytogenes, Vibrio parahaemolyticus, V. cholera, and Staphylococcus aureus) in aquatic products were

used as targets to evaluate the sensitivity, specificity and repeatability of this method. The detection sensitivity

of this method can reach 103CFU/mL, and it is no different from the specificity of fluorescence detection.

The coefficient of variation CV value of the hybridization repeatability results of different points on the same

chip and different batches of chips were also less than 15%. The sensitivity, specificity, and reproducibility

of the four visual gene chip detection methods for pathogenic bacteria in aquatic products all show good

performance, which has practical promotion significance in the detection of pathogenic bacteria in aquatic

products.

1 INTRODUCTION

Food safety has become a widespread and far-

reaching social issue. With the development of

society and people’s concerns regarding health, the

safety and health of aquatic products is receiving

increasing attention. Consumers and government

authorities are paying more and more attention to the

quality and safety of aquatic products, and the safety

and quality management of aquatic products has

become an important factor in ensuring food quality

and promoting social stability (Gao et al., 2007).

China is one of the largest producers of aquatic

products in the world. Over the past 20 years, total

aquatic production has continuously grown, and

China’s aquatic production reached about 49 million

tonnes in 2011, accounting for 35% of the world’s

aquatic production, ranking it first in the world. In

particular, aquaculture production accounted for

more than 70% of the world total (Weng et al., 20211,

Shuai et al., 2011). However, the detection rate of

Listeria monocytogenes in salmon in China was 5–

7%, of Vibrio parahaemolyticus in elephant mussels

was 9–10%, and of V. parahaemolyticus in jumbo

crab was 7–8%. Vibrio cholerae, a national Class I

controlled infectious disease, had a detection rate of

about 3%, and pathogenic Escherichia coli and

Salmonella were also detected at high rates (Wang

and Tao, 2009, Wang et al., 2008, Jin et al., 2008).

Internationally, the European Union, the United

States, Japan, and others have strengthened their

health and safety testing measures for imported

aquatic products or increased their detection

programs. For example, the European Union

stipulates that the four pathogenic Vibrio species with

the highest risk - V. parahaemolyticus, V.

alginolyticus, V. cholerae and V. vulnificus – should

not be detected in shrimp and fish products, and other

aquatic products also have similar requirements for

corresponding Vibrio species. Because the quality of

China’s exports of aquatic products often varies and

can exceed the required acceptable pathogen levels of

importing countries, products are often rejected,

creating a series of “green barriers”. Such measures

result in greater challenges to China’s aquatic

products export trade (Bobrow et al., 1989).

Because chilled and fresh aquatic products are

not easy to preserve, a rapid and sensitive detection

method is urgently needed to protect the interests of

businesses and the health of consumers. The existing

methods of detecting pathogenic bacteria in aquatic

products in China, whether for import or export

industry standards, or national standards, take a long

time. Negative results need at least 3–4 days, and

positive results need 5–10 days. Existing detection

Lu, L.

Evaluation of Method of Visual Gene Chip Detection for Four Pathogenic Bacteria in Aquatic Products.

DOI: 10.5220/0011595700003430

In Proceedings of the 8th International Conference on Agricultural and Biological Sciences (ABS 2022), pages 50-55

ISBN: 978-989-758-607-1; ISSN: 2795-5893

methods such as immunochromatography, ELISA,

and PCR can only detect one kind of foodborne

pathogenic bacteria at a time. Many experiments are

needed to identify or investigate a suspicious sample,

and the operation is cumbersome and time-

consuming, and cannot meet the needs of multiple

detection (Meany et al., 2011, Deng et al., 2011, Qi et

al., 2010). To address this need, the author has

combined tyramine signal amplification technology

and nanogold-labeled silver staining technology. A

visualized gene chip detection method was

established for simultaneous detection of four

pathogenic bacteria in aquatic products: L.

monocytogenes, V. parahaemolyticus, V. cholerae,

and Staphylococcus aureus.

2 MATERIALS & METHODS

Strains: The standard strains used in this experiment

were selected by the Academy of Military Medical

Sciences: L. monocytogenes, V. parahaemolyticus,

V. cholerae, and S. aureus. The strain names and

standard strain numbers are shown in Table 1.

Table 1: Experimental standard strains and their numbers.

Strain Bacteria numbe

Listeria monocytogenes

54003, 54005, 54006,

54007, 7644

Vibrio parahaemolyticus

20502, 20506, 20507,

20511

Vibrio cholerae O139 M045

Staphylococcus aureus

26001, 26111, 26113,

13565, 27661

Clinical samples: The 10 positive reference

strains used in our laboratory were positive strains of

imported aquatic products. They were identified as

pathogenic bacteria of L. monocytogenes, V.

parahaemolyticus, V. cholerae and S. aureus by

conventional culture methods. The samples were

cultured in enrichment solution for 3 h before use.

Instruments and reagents: The instruments used

included an iCycler PCR instrument (Bio-Rad Co.),

GenePix 4000B scanner (Axon Co.), PixSys 5000

spotter (Cartesian Co.), and Model 8909 DNA

synthesizer (ABI Co.). The PCR-related reagents

were purchased from Bao Biological (Dalian) Co.

Ltd., gene chip substrate from CEL Associates Co.,

Cy3 fluorescent dye from Amersham Co., tyramine

signal amplification-Cy3 reagent from PE Co., and

streptavidin–HRP and coupling buffer from Sigma

Co.

Evaluation of gene biochip specificity: The

standard strains of the target pathogenic bacteria and

the standard strains of related proximate and distant

genera were selected and amplified by multiplex PCR

with universal primers. They were then hybridized

with a variety of pathogenic detection probes

obtained by screening, and the specificity of each

probe on the gene chip was systematically evaluated.

Evaluation of gene chip sensitivity: The

sensitivity standards of various pathogenic bacteria

were provided by the China Institute of Drug and

Biological Product Identification and the Institute of

Microbiology and Epidemiology of the Academy of

Military Medical Sciences. The standards were

diluted with saline in a 10-fold gradient, and the DNA

processed with reference to the genomic DNA

extraction procedure.

Evaluation of gene chip repeatability: Three

batches of L. monocytogenes were randomly selected

from different batches of biochips for repeatability

tests. Then three chips were randomly selected from

three different batches of chips, and the detection

results obtained by multiplex PCR amplification and

gene chip hybridization were statistically analyzed to

evaluate the repeatability of the chips.

Preparation of fresh shrimp liquid contaminated

with L. monocytogenes: Fresh shrimp detected by

conventional culture methods in our laboratory were

pulped by apparatus, and 1 mL of serially diluted L.

monocytogenes DNA diluent (10

–10

CFU/mL) was

added to each 0.1 g of fresh shrimp pulp to make a

final concentration of 10

–10

CFU/mL/0.1 g

suspension of fresh shrimp pulp. Then 9 mL of 75%

ethanol was added, mixed, and centrifuged at 100×g

for 1 min to remove large particles. The supernatant

was transferred to a new centrifuge tube and

centrifuged at 10,000×g for 10 min. Then the

supernatant was carefully aspirated, sterilized saline

was added for one washing, and DNA extract added

to the final precipitate, boiled for 10 min, and

centrifuged. Of the supernatant, 8 μL was used for

PCR detection. Preparation of sensitivity simulation

samples of V. parahaemolyticus, V. cholerae, and S.

aureus: V. parahaemolyticus, V. cholerae, S. aureus

sensitivity simulation samples were prepared

referring to the previous section.

Evaluation of Method of Visual Gene Chip Detection for Four Pathogenic Bacteria in Aquatic Products

3 RESULTS

3.1 Specificity of Gene Biochip

Detection

To examine the specificity of the method for the

detection of the four pathogenic bacteria, the positive

and negative references provided in Table 1 and from

the Academy of Military Medical Sciences were

compared. The results of gene biochip hybridization

showed that the probes for the four pathogenic

bacteria were highly specific under the above

optimized reaction system (Figure 1). For the other

non-target pathogenic bacteria, no fluorescent signal

appeared at any probe position except for the positive

signal of the internal control probe, while no

fluorescent signal appeared for the negative probe or

the blank control (Azizipour et al., 2020).

3.2 Determination of Cutoff Value

Cutoff value is the criterion to determine whether the

fluorescence signal value of a gene chip is positive or

not. In this experiment, the cutoff value of the probes

was calculated and determined based on the

specificity evaluation. For each probe, a positive

strain, a negative strain, and a blank were selected for

gene chip hybridization. The hybridization

fluorescence signal value of the positive strain was

much higher than the background value of the

negative strain and the blank control hybridization

(Figure 1). Through repeated experiments, we

determined the background statistical average of the

negative strain and the blank control + 2 standard

deviations as the cutoff value of each probe (Table 2).

Table 2: Cutoff values of four aquatic product pathogenic

bacteria probes.

Bacterial name

Fluorescenc

e mean of

negative

bacteria and

blank

control

Standard

deviation

Cutoff

value

Listeria

monocytogenes

485.25 24.89 535.03

Vibrio

parahaemolytic

586.75 34.13 655.01

Staphylococcus

aureus

696.27 52.60 801.47

Vibrio cholerae

O139

467.6 36.15 539.9

Vibrio cholerae

non-O139

278.625 30.28

339.18

Internal control 221.16 22.90 266.96

Figure 1: Specificity detection results.

ABS 2022 - The International Conference on Agricultural and Biological Sciences

3.3 Sensitivity of Pure Culture

Pathogenic Bacteria

In this experiment, the sensitivity of pure cultures of

L. monocytogenes, V. parahaemolyticus, V. cholerae,

and S. aureus were evaluated separately. The

sensitivity of the detection was determined according

to the cutoff values of the probes (Table 2) after the

initial concentrations of the above target bacteria

were diluted with saline in a 10-fold gradient, directly

boiled and lyse, PCR amplified, and hybridized. The

results are shown in Figures. 2–5 (Park et al., 2018,

Bunin et al., 2020).

Figure 2: Sensitivity of detection of Listeria monocytogenes.

Figure 3: Sensitivity of detection of Vibrio parahaemolyticus.

Figure 4: Sensitivity of detection of Vibrio cholera.

Evaluation of Method of Visual Gene Chip Detection for Four Pathogenic Bacteria in Aquatic Products

Figure 5: Sensitivity of detection of Staphylococcus aureus.

Figure 6: Gene biochip repeatability evaluation.

The sensitivity of all four pathogenic bacteria

reached 10

CFU/mL (Figures 2–5), while L.

monocytogenes and S. aureus corresponding to 10

CFU/mL showed fluorescent signals in the

hybridization plot, but the signal value was lower than

the cutoff value of the probe of the bacteria, so it

could not be taken as a positive result. Meanwhile, we

observed from Figure 4 of hybridization of LPSgt and

ctxA genes of V. cholerae O139 that when

hybridizing one gene alone, the fluorescence signal

value of each gradient was higher than the value of

simultaneous hybridization. Apparently two genes

hybridized at the same time affected each other,

probably because the annealed PCR products

hybridized with each other, reducing the amount of

hybridization with the corresponding probe, but the

statistical analysis of simultaneous hybridization

showed that sensitivity reached 10

CFU/mL (Dou et

al., 2019).

3.4 Repeatability of Gene Biochip

Detection

Listeria monocytogenes was selected to evaluate the

repeatability of the gene biochip. The PCR

amplification and gene biochip hybridization were

performed with 10

CFU/mL of the pathogenic

bacteria, and a negative control without a template

was set up. The experiment was repeated five times

and intra- and inter-slice repeatability were

calculated, thus assessing the repeatability of this

method of detection. The results of the experiments

are shown in Figure 6, and the statistical analysis

results are shown in Table 3. For the single-amplified

L. monocytogenes probe, the coefficients of variation

(CV) of the repeatability of different spots on the

same chip were less than 15%, and the CVs of the

hybridization repeatability results of different batches

of spot systems of the chip were also less than 15%,

indicating that repeatability of detection was good

(Park et al., 2018).

Table 3: Statistical results of gene biochip repeatability

evaluation.

Repeatability

Number of

experiments

Internal

control

probe 1

Listeria

monocyt

ogenes 1

Intra-Slice

repeatability

V (%)

1 8.31 6.38

2 7.23 9.46

3 5.29 8.33

4 9.33 8.74

5 7.45 9.12

Inter-slice

repeatability

CV (%)

7.82 8.65

ABS 2022 - The International Conference on Agricultural and Biological Sciences

4 DISCUSSION

Cutoff value is a criterion to determine whether a

hybridization fluorescence signal is positive or not. In

this experiment, the background values of positive

fluorescence signal value, negative fluorescence

signal value, and blank control were statistically

analyzed through multiple repetitions, and the

corresponding cutoff value determined for each probe

(Table 2). The cutoff value is not only used to judge

the hybridization results, but also provides a reliable

basis for the evaluation of sensitivity. The sensitivity

of this experiment was respectively evaluated for L.

monocytogenes, V. parahaemolyticus, V. cholerae,

and S. aureus, and the sensitivity reached 10

CFU/mL. This experiment evaluated the

reproducibility with L. monocytogenes and showed

that the CV of inter- and intra-slice reproducibility of

this gene chip was less than 15%, indicating that the

method was accurate, reliable, and repeatable.

Therefore, the results of this method for the

evaluation of gene chip detection for the visualization

of the four aquatic pathogenic bacteria showed the

high specificity of each probe, the intra- and inter-

slice CV was less than 15%, the repeatability was

good, and the sensitivity of the four pathogenic

bacteria reached 10

CFU/mL. Therefore, the

detection method can be used for sensitive and high-

throughput detection of four kinds of aquatic products

pathogens, which provides fast and accurate detection

technology for improving the pathogenic bacteria of

imported and exported aquatic products, and is of

great significance for ensuring the quality and safety

of imported and exported aquatic products.

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Evaluation of Method of Visual Gene Chip Detection for Four Pathogenic Bacteria in Aquatic Products