real time pcr for the detection of salmonella in food

List of Contents

ADSUVACE ccc cc een ec ene EEL LE EEEE DLE EELEACLEEDLE CLES CLECLLEECLELLEAELEDLEEELEESLIEECLEELEEEOEELEGEEEEtEEOREaEE 3

1 Introduction L0 u nh 2e ree 3 2 Materials and method chu Hruea 5 2.1 Materials L 0.0 nh HH2 2 na 5 2.1.1 Tools 2n 1212221110121 21121 na 5 2.1.2 Equipments nga 7 2.1.3 Overview of Salmonella real time PCR kit (Nam Khoa biotek' kit) S2 H2 n1 n1 n1 re § 2.2 Methods ch HH 2 tre 9 3 Result and discussion L0,” 2222222222 terns 15 4 Conclusion n2 21 2 H211 are 19 5 Ref©er©enc©S ch nh HH HH tra 19

Abstract

Salmonella is a bacterium that can cause serious foodborne illnesses, particularly from contaminated meat products Real-time PCR is a commonly used technique for detecting Salmonella in food samples However, after conducting an experiment to check

for Salmonella in pork and chicken meat, it was found that the efficiency of the real-time PCR was

affected by the long time operations after adding the Master mix This resulted in false-positive or negative results, leading to interference in the accuracy of the test This report highlights the importance of optimizing the PCR conditions to avoid false results and ensure the accuracy of Salmonella detection in food samples

Introduction

Salmonella belongs to the Enterobacteriaceae family and is a Gram-negative, rod-shaped, facultative anaerobic bacterium The genus Salmonella is an extremely polymorphic and diverse = group,

comprising approximately 2,500 serovars (Brenner,

Villar et al 2000) Salmonella is the type of bacteria that can cause a disease in the digestive tract of humans and animals called Salmonella infection Acute diarrheal disease remains a major public

health issue Salmonella spp infection is one of the

leading causes of acute diarrheal disease despite the preventive measures implemented (Popa and Papa 2021) Food sources of Salmonella included mainly milk, eggs, meat (poultry, beef) vegetables, and fresh fruits (Almeida, Cerqueira et al 2013),

particularly those of animal origin and those in

contact with sewage pollution

In many countries, it is the leading cause of food- borne infections and outbreaks (Tirado and Schmidt 2001), including Viet Nam Vietnamese National Standards (TCVN: 7926-2008 Meat and meat products and TCVN 6040: 2007 Microbiology of food and animal feed) required amount Salmonella in 25 g of food is 0 However, in a study about Antimicrobial

3

susceptibility of Salmonella spp isolated from raw meats at traditional markets in Ho Chi Minh city showed that the prevalence of Salmonella spp is 42.37% In which, the infection rate in each sample group was 50.00% for pork, 49.62% for chicken meat, respectively Salmonella bacteria isolated from

fresh meat retailed at traditional markets in Ho Chi

Minh City have a high rate of multi-resistant, antibiotic resistance genes, especially ESBL- producing strains genes encoding resistance (VG, Van Hai et al 2021)

Vietnamese National Standards (TCVN: 7926-2008 Meat and meat products and TCVN 6040: 2007 Microbiology of food and animal feed) required amount Salmonella in 25 g of food is 0

Traditional culture methods always involve multiple culturing stages combined with test steps that take 5 to 7 days (ISO 6579:2003) The main limitation of this method is that it is time-consuming, low in sensitivity, and labor-intensive, which the food stocks being analyzed are forbidden to be sold and makes it

unsuitable for routine testing of large numbers of samples Therefore, the inspection of food for the

presence of Salmonella has become routine all over

the world (Malorny, Paccassoni et al 2004)

The rapid, cost-effective, and automated diagnosis of food-borne pathogens throughout the food chain continues to be a major concern for the food industry and public health Because of these requirements, the PCR became a powerful tool in microbiological diagnostics during the last decade (Sachse 2003) Polymerase chain reaction, or PCR, is a DNA cloning reaction based on thermal cycles PCR uses repeated cycles of heating and cooling to make many copies of

a specific region of DNA First the temperature is

raised to near boiling, causing the double-stranded DNA to separate or denature into single strands When the temperature is decreased, short DNA sequences known as primers bind or anneal to

complimentary matches on the target DNA

sequence The primers bracket the target sequence to be copied At a slightly higher temperature, the

enzyme taq polymerase binds to the prime

sequences and adds nucleotides to extend the

4

second strand This completes the first cycle In subsequent cycles, the process of denaturing, annealing, and extending are repeated to make additional DNA copies After three cycles, the target sequence defined by the primers begins to accumulate After thirty cycles, as many as a billion copies of the target sequence are produced from a single starting molecule

Similar to PCR, real-time PCR is also a DNA cloning technique based on thermal cycles Real-time Polymerase Chain Reaction (PCR) is the ability to

monitor the progress of the PCR as it occurs (i.e., in

real time) Data is therefore collected throughout the

PCR process, rather than at the end of the PCR This

completely revolutionizes the way one approaches PCR-based quantitation of DNA and RNA In real-time PCR, reactions are characterized by the point in time

during cycling when amplification of a target is first

detected rather than the amount of target accumulated after a fixed number of cycles The higher the starting copy number of the nucleic acid target, the sooner a_ significant increase ¡in fluorescence is observed In contrast, an endpoint assay (also called a “plate read assay”) measures the amount of accumulated PCR product at the end

of the PCR cycle Diagnostic real-time PCR for the

specific detection of Salmonella in foods ¡is increasingly being used as a rapid and reliable tool for the control of contaminated samples along the food production chain (Malorny, Paccassoni et al 2004)

The aim of this study to detect and quantification of amount of Salmonella in pork and chicken which taken from market Especially is in Vietnam, the risk of infection is particularly high due to unsafe

slaughtering methods, which have led to a high

incidence of Salmonella in both chicken and pork meat Additionally, the presence of antibiotic- resistant strains of Salmonella in these meats exacerbates the public health concern

2 Materials and method 1 Materials

- 25g chicken meat - 25g ground pork meat

- 6.75g Buffered Peptone Water (BPW)

Salmonella with Fam and Hex filter channel, and dNTP (Deoxyribonucleotide triphosphate - the

canonical substrates that polymerases Uuse to

3) “SM C [-] is TE 1X

4) DEPC is distilled water treated

2 Methods

Prepare the raw materials

BPW liquid: weight 3.375g BPW powder on silver paper Suspend 3.375g of the powder in 225ml of

distilled water into each schott bottle Mix well and

shack frequently until completely dissolved

iy ane

(777777777 ile 0 4 ⁄⁄

2⁄2

2⁄7

LLP

Picture 2: BPW liquid, laboratory forcep,

pipette tips, eppendoft for sterilizing step

After sterilize, using the laboratory forcep to grip

25g of chicken sample and 25g of ground pork meat samples into each stomacher bag

Picture 3: 25g of chicken sample and 25g of ground pork

meat samples into each stomacher bag

For pre-enrichment, add sample to Buffered Peptone Water by pouring 225ml BPW solution into each bags

Picture 4: Pouring the BPW solution into

stomacher bag

11

Picture 5: Pork and chicken sample in stomacher bags after pouring the BPW solution

- Putting into the slap-type homogenier to blend the

samples This step helps lysing of cells to form one

homogeneous suspension

Picture 6: Putting samples into the slap-type homogenier

- Tie the bags

Picture 7: After tying the bags

- Incubate at 37 + 1°C for 24 hours before transfer to selective enrichment media

12

Picture 8: Incubating samples at 37 + 1°C for 24 hours

¢ Real-time PCR steps

Step 1: Aspirated 1ml the pork and chicken

enrichment solution in each Eppendoft tube, caped the tubes

Picture 9: Aspirating Iml the pork and chicken enrichment solution in each Eppendoft tube

Picture 10: Sample solution in each Eppendoft tube

Step 2: Followed by cold centrifugation at

13000RPM in 5 minutes

13

Picture 11: Putting pork Eppendoft and chicken Eppendoft in centrifuge

Picture 12: Pork Eppendoft and chicken Eppendoft after centrifuging Chicken sample on the left and pork sample

on the right

Step 3: Removed supernatant, retained residue

Picture 13: After removed supernatant, retained residue

Step 4: Performed under the flame of alcohol Then

added 100um DEPC in each Eppendoft

14

Picture 14; Adding 100um DEPC in each Eppendoft

Step 5: Shacked gently to mix well

Step 6: Used the dry block heater to provide a hygienic heating incubation at 90°C for 10 minutes

Picture 15: Incubation at 90°C for 10 minutes

Step 7: Centrifugation at 13000RPM in 5 minutes

Step 9: Used micropipette to distribute 15m ““SM

TQPCR master mix in each 0.1ml Eppendoft tube

15

Picture 17: Adding Master mix

Step 10: Aspirated 5um DNA extraction of pork and

chicken samples in each 0.1ml Eppendoft tube which already contained prepared the “SM TQPCR master mix, caped the tubes

Picture 18: Extraction of pork and chicken samples

in each 0.1ml Eppendoft tube

Step 11: Centrifuge by spin-down centrifuge for a

few second to concentrate all the drops adhering to the walls of the test tube to the bottom

Then put tubes in Thermo cycler and run PCR

analysis

Set up the the thermal cycle of real-time PCR reaction using TaqMan probe has 2 stages: 1) 1 cycle (denature) at 95°C for 15 minutes 2) 45 cycles and 2 temperature steps (pairing and stretching) at 95°C for 15 seconds and 60°C for 60 seconds

Result and discussion

At the beginning of a PCR run, the amount of PCR

product is low, indicating very little fluorescence

since amplification is just beginning The emitted fluorescence signal is not strong enough for the

16

machine to measure, the signal line will be horizontal (called the background signal) This phase of the reaction (usually occurs between cycles 0 and 15) is known as the stationary baseline phase It happens due to dna multiplication by adding mastermix However, multiplying by insufficient amount When the number of copies of DNA is large enough, the machine will begin to record the emitted fluorescence signal, and the signal will increase with each thermal cycle, this phase is called the log phase (cycles 16-25) In this phase, there is a very

important parameter that is the threshold period (C;), this is the period at which the fluorescence signal in the PCR tube begins to cross the background signal

line In real-time PCR reaction, depending on the

initial sample, C, will appear sooner or later If a large amount of DNA template is present at the start of the reaction relatively few cycles are required to

generate enough product to give a signa above background thus the reaction will have a low baseline phase or Ct will appear early Conversely

small amounts of DNA template generate larger

baseline phase or C; will appear late

At a certain period, the reaction is exhausted, microorganisms used up dNTP and the Taq

polymerase enzyme is no longer effective, leading to the signal not to continue to increase, this stage is called the plateau phase

17

Fig 1: Temperature profile for the RT and PCR phases

In the PCR, the temperature changes according to two cyclic values (95 and 60 °C) There are 45 cycles (45 rounds of amplification) The time for Thermal cycle started at 16.40 minutes and the end at 01:23:20 hours which occurred in 66 minutes 83 seconds

As shown in Fig 2; Fig 3; Fig 4; and Fig 5, the real- time PCR combined with guided culture protocol could only detect Salmonella at the corresponding

fluorescent channel (HEX and FAM for pork and

chicken sample) The plot resembled a sigmoidal amplification pattern, as indicated by the blue line for dye fam and hex of pork sample, and the green for of dye fam and hex of chicken sample

18

Fig 2: Fam dye of pork sample

Fam dye of pork sample was positive There may be

small spikes in the curve known as “noise”

Therefore, the results were still considered noisy but not too much In generel, the presentation of the phases could be acceptance

Fig 3: Hex dye of pork sample

In Hex dye of pork sample, the cycle number when

the threshold intersects the amplification plot were

18.85 The sample was considered positive (< 29) and it is a strong positive reaction indicative of abundant target nucleic acid in the pork sample The

low Ct value, the more PCR product that were 19

present due to it took fewer PCR cycles for that product to be detected over the background signal

Fig 4: Fam dye of chicken sample

The sample was considered false negative based on the Fam dye There were still small spikes in the curve The noise of sample occurred due to the

method of extraction, as well as the timing of sample

collection

Fig 5: Dye Hex of chicken sample

20

In Hex dye of chicken sample, Thermal cycle was negative The sample was considered negative because the Real-time PCR could not be identified C: Tube of chicken sample did not give amplification of both Fam and Hex dye, the sample could be inhibited and inconclusive This result occurs due to the operation after adding Master mix was not fast and accurate

sample el al

Hex [+] Salmonell a

Hex [-] sample

Table 1: Results tested Salmonella on pork and chicken

sample by the PCR method

Both pork and chicken samples gave the incorrect results due to The external contamination of PCR products Meaning that the bacteria may be present in the sample but the test result may show that it is not It is important to ensure that the sample is not inhibited during manipulation to obtain accurate results

Conclusion

In this study, a real-time PCR was developed for the detection of Salmonella in food The method consists

of a preenrichment step of the sample in BPW done

overnight followed by an extraction-purification step for the bacterial DNA The DNA is finally analyzed by the real-time PCR assay for the presence of

Salmonella DNA

PCR is a very powerful and extremely sensitive

amplification technique, but there is always the peril

that a tiny amount of contamination of the DNA

target may lead to false-positive or negative results Many factors can affect the efficiency of a given RT- PCR, particularly the choice of sample and the method of extraction, as well as the timing of sample collection Individuals experiencing prolonged viral

21