Khóa luận tốt nghiệp Công nghệ sinh học: Evaluating ability of Detection of African Swine Fever Virus (ASFV) in swine of the trial kit TraceDetectTM qPCR asfv

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESEVALUATING ABILITY OF DETECTION OF AFRICAN SWINE FEVER VIRUS ASFV IN SWINE OF THE TRIA

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

EVALUATING ABILITY OF DETECTION OF AFRICAN SWINE FEVER VIRUS (ASFV) IN SWINE OF THE TRIAL KIT

MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCES

Dr DINH XUAN PHAT NGO THI CAM PHIN

Thu Duc City, 8/2023

I would like to express my sincere gratitude towards the Board of Nong LamUniversity Ho Chi Minh City and the Board of Faculty of Biological Sciences forcreating the most favorable conditions for me to finish this study

I would also like to thank all the teachers of the Faculty of Biological Scienceswho have transferred valuable knowledge during my years

And especially, I would like to express my deep gratitude to Dinh Xuan PhatPh.D for wholeheartedly teaching, guiding and supporting me during the thesis process

Many thanks to Ms Nguyen Thi Mi Mi, Mr Le Thanh Binh, Mr Nguyen Thanh

Canh, Ms Tran Nguyen Minh Thanh, Ms Chau Ngoc Huynh Nhu and other members

of Gene Technology Laboratory BIO313 for always helping, sharing, and supporting

me to reach my goals

Finally, I would like to thank my family for always loving and trusting me And

my friends have accompanied me on this journey

Sincere

CONEIRMATION COMMITMENT

My name is Ngo Thi Cam Phin, Student ID: 19126132, Class: DH19SHD,Faculty of Biological Sciences, Nong Lam University Ho Chi Minh City I guaranteethat: This research was conducted by myself, the data and information in the researchare completely honest and objective I take full responsibility before the Council forthese commitments

Thu Due City, September 2023

Student’s signature

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The topic research “Evaluating ability of detection of African Swine Fever

Virus (ASFV) in swine of the trial kit TraceDetectTM qPCR ASFV” was performed to Virus (ASFV) in swine of the TraceDetectTM qPCR ASFV kit” was performed to check

effectiveness of trial kit through evaluating specificity and limit of detection in fieldsamples In this study, limit of detection of trial kit was 1 copy/uL and the trial kit onlyamplified and specific for ASFV and not amplified for unrelated DNA/RNA Standardcurve of trial kit was y = -3.5854x + 33.722 with parameters of Slope = -3.5854; andEfficient % (E%) = 90.06% The diagnostic sensitivity and specificity values of the trialkit were determined to be 100%; besides, the predicted positive value and the predictednegative value were 100% The coefficient of variation between replications on the testkit ranged from 0.43% to 7.39% In summary, the study showed that the trial kit was

competent to be used in diagnosis of ASFV in field samples

Keywords: African Swine Fever Virus, ASFV, trial kit, real-time PCR

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TÓM TẮT

Đề tài “Đánh giá khả năng phát hiện African Swine Fever Virus (ASFV) trên lợn của bộ kit thử nghiệm TraceDetectTM qPCR ASFV” được thực hiện dé kiểm tra hiệu quảcủa bộ kit thử nghiệm thông qua việc đánh giá độ đặc hiệu và giới hạn phát hiện trên

mẫu thực địa Trong nghiêm cứu này, giới hạn phát hiện của bộ kit thử nghiệm là 1

copy/uL và bộ kit thử nghiệm chỉ khuếch đại và đặc hiệu với ASFV và không khuếch

đại với DNA/RNA không liên quan Đường chuẩn của bộ kit thử nghiệm là y = -3.5854x

+ 33.722 với các thông số Độ dốc = -3.5854; và Hiệu quả khuếch đại (E%) = 90.06%.

Các giá trị độ nhạy chuẩn đoán và độ đặc hiệu chan đoán của bộ thử nghiệm được xác định là 100%; bên cạnh đó, giá trị dự đoán dương và giá trị dự đoán âm là 100% Hệ số

biến thiên giữa các lần lặp lại trên bộ thử nghiệm dao động từ 0.43% đến 7.39% Tóm

lại, nghiên cứu cho thấy rằng bộ thử nghiệm có thé được sử dụng dé chân đoán ASFV trên các mẫu thực địa.

Từ khóa: African Swine Fever Virus, ASFV, kit thử nghiệm, realtime PCR

IV

TABLE OF CONTENTS

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LIST OF ABBREVIATIONS nseiniieeuiioeoodsnodisdoooostvetasslkigSgsglbsBSsssiSsS0008003081490380 Vil

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De BDin Bextra CLOT) NA eo csccctincnaewtt ramet etnias ttn eet ef see 13.3.3 Realtime PCR feRAGHDÍlicsssessaendidisdieBS20143830095504.1815613181500003800138E610S0141848408309860E 193.3.4 Determination of detection ÏITIL - 5-2222 * 22+ 3z £+2EE+zE+zEseeezkeereerrrrseese 1933.5 4 estInơ:arialyfl@đ] speciietty xs ccc tise n 06x G0 b2 1b sec C06 ko E022 022 x<e 193.3.6 Determination of detection limits on field sampÌes - ¿55555 +<+<£+<x<+s 203.3.7 Check accuracy 1n ASFV def€CtIOH SG 22222212211 151 1211121111111 E11 1 re 203.3.9 Evaluation of the stability of the trial kit for the detection of ASFV 213.4 Data processing na 21CHAPTER 4 RESULTS AND DISCUSSION 2-2-2 22VÌ; RESU TS sceeorermrenss semen ery s taunt ereernecivemneainnyssboer ee oerrmmerrererneemeeem eereeets 224.1.1 Determination of detection limit +52 ++s++*£+zEs+eezzErxerererrrrrrerrrrrrrre 22

4.1.2 Testing analytical SD€CIÍÍC1EY - G22 2 2221221223121 15312 111112 111111111211 11 xe 23

4.1.3 Determination of detection limits on field sampÏes - 5 =+=+<c+<c+s 244.1.4 Comparative values between the trial kit and Reference KIf 25I1 -2-.ISGUISSTGTiSsxxssessbsisooEibsuesadlbssaiageduoisiergDadtsEisgriiigsglsseEngusoulizcuossdkicezosuEsbiessilSđoassiSfrbsrzodishrsnzed 28Chapter 5 CONCLUSION AND RECOMMENDA TIONS -.e 30B90) 5.2 Recommendation 1n ÓREFERENCE ngán giáo in cece yr caterer tase a tte ce 31BBE NOS cesses ee ni a rete Bw es ee eae 0

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: African Swine Fever

: African Swine Fever Virus

: Coefficient of Variation

: Deoxyribonucleic Acid

: Gross Domestic Product

: Limited of Detection

: Minor Groove Binder

: Negative Predictive Value

: Office International des Epizooties

: Polymerase Chain Reaction

: Positive Predictive Value

: Sensivity

: Specificity

Vii

LIST OF TABLES

Page

Table 2.1 Main lesions observed in the different forms of ASE - 7

Table 3.1 Evaluate the negative and positive results of the trial KIt - 10

Table 4.1 Results of three replicates for serial 10-fold dilutions of ASFV DNA 22

Table 4.2 ASFV detection results on field samples of 2 Real-time PCR kIfs 25

Table 4.3 The trial kit's ability to detect ASFV in 2 samples platforms 26

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The graph shows the stages and parameters of Real-time PCR 13

Determine LOD of the ASFV TracdetectTM qPCR ASFV Kt 22,

The standard curve of ASFV plasmid DNA - 5555555<<+<<<<52 23

Result Real-time PCR to testing analytIcalL - - << << <++ssssscesss 24The results of real-time PCR of 10 samples after 4 times 10-fold 25The result of detection ASFV of both kits in 2 field samples 27The stability of the trial kit of 29 positives with ASFV samples 28

1X

CHAPTER 1 INTRODUCTION

1.1 Introduction

African Swine Fever (ASF) 1s one of the perilous diseases caused by AfricanSwine Fever virus in pigs The ASF had high infectivity and mortality (up to 100%)(Galindo et al., 2017; Li et al., 2022) ASF first appeared in Kenya in the 1920s, Europe

in 1957, the Caucasus region, and southern Russia in 2007 (Cwynar ef al., 2019;Sanchez ef al., 2012) According to the OIE, China was the first country that reported

an outbreak of ASF in East Asia in August 2018 (Mighell and Ward, 2021) It rapidlyspread to most provinces in China within a short period of time and adversely affected

the swine industry (Tao ef a/., 2020) Furthermore, ASF had been reported in other Asian

countries since the end of 2018 (Mighell and Ward, 2021)

According to a Department of Animal Health report, African Swine Fever Viruswas first detected in Vietnam in February 2019 After that, the disease spread on a largescale within 5 months, causing heavy damage to pig production in our country In April

2020, the ASF epidemic in Vietnam appeared and continued to spread in 2021 and 2022(Thinh Nguyen-Thi ef a/., 2021) Pig production occupied 60% of the total livestockoutput in Vietnam The ASF outbreak and presence in the long term caused severeeconomic losses among farmers and posed adverse impacts on the pig industry in ourcountry, leading to a decrease in the gross domestic product (GDP)

Because of the seriousness of this virus, early detection of the virus in the sample

is essential To meet this market demand, real-time PCR reaction kits were created and

TraceDetectTM qPCR ASFV Kit is one of them Currently, to simplify the process, many

products on the market have a collection of key components for real-time PCR reactions,called real-time PCR kits To establish a sensitive, specific assay for the detection andquantitation of ASFV, testing and evaluating the kit at the research stage is important.Therefore, "Evaluating ability of detection of African Swine Fever Virus (ASFV) on the

trial kit TraceDetectTM qPCR ASFV" was performed.

1.2 Objective

Evaluating ability of detection of African Swine Fever virus (ASFV) on the trial

kit TraceDetectTM qPCR ASFV.

1.3 Research contents

Content 1: Evaluate the parameters (specificity and limit of detection) of

TraceDetectTM qPCR ASFV kit.

Content 2: Successfully comparison and evaluation the ability to detect ASFV

on different field samples with a trial kit

CHAPTER 2 LITERATURE REVIEW

2.1 Introduction of African Swine Fever virus (ASFV)

2.1.1 Structural property

ASFV is a large, icosahedral, linear double-stranded DNA virus and the only

virus classified under the Asfarviridae family and Asfivirus genus (Dixon ef a/., 2005).The virion has a multiple-layer structure, including the envelope, capsid, inner envelope,core shell and nucleoid from outside to inside (Heath et a/., 2001; Xian and Xiao, 2020)

as shown in Figure 2.1

2.1.1.1 Outer envelope

The outer envelope is the outermost layer of ASFV virion which was acquiredfrom the host cellular membrane during budding The outer envelope is the outermostlayer of ASFV which was acquired from the host cellular membrane during budding

The morphology of the external envelope is by the budding process for virus exit from

the cell (Breese and DeBoer, 1966) p12 was seen as a protein that promotes theadsorption of virus particles on host cells as an outer envelope protein by binding to

specific receptors on the host cell membrane to mediate ASFV entry (Alejo ef a/., 2018)

2.1.1.2 Capsid

The capsid is composed of about 2,760 pseudo-hexameric capsomers and 12pentameric capsomers Protein p72 was seen as with the major capsid protein ofiridoviruses, phycodnaviruses and mimiviruses, which also had icosahedral capsidssurrounding an inner envelope (Iyer ef a/., 2001) Three p72 protein molecules adopting

a double jelly-roll structure form one pseudo-hexameric capsomer, and another fivepenton proteins could construct a pentameric capsomer (Wang ef a/., 2019) In addition

to p72, protems such as pB438L and pE120R were constitutive proteins of the viralcapsid The protein pB438L is needed for the capsid to form its peaks (Alejo ef al.,2018)

2.1.1.3 Inner envelope

The third layer is a 70-A thick lipid bilayer membrane divided from the

endoplasmic reticulum (ER) (Alejo e/ al., 2018) The membrane proteins p54 (J M.Rodriguez ef al., 2004), p17 (Suarez et al., 2010), and pE248R(1 Rodriguez et al., 2009)are constituent of this layer

2.1.1.4 Core shell

The core shell was a thick protein layer of about 30 nm and 180 nm diameter(Alejo et al., 2018; Revilla et al., 2018), and was defined as an independent domain of

the virus core encompassing the central nucleoid (Andrés ef al., 1997) Two kinds of

virus polyprotein precursors, pp220 and pp62 were broken down into many matureproducts through viral protease (pS273R) to form the core shell

2.1.1.5 Nucleoid

The innermost virus particles are nucleoids ASFV genome 1s double-strandedDNA approximately 170 to 194 kbp and encodes 150 to 170 open reading frames(Cackett et al., 2020) The nucleoid contains the viral genome and nucleoproteins such

as the DNA-binding protein pA104R and protein p10, which is similar to the like proteins of bacteria (Andrés ef al., 2002; Muñoz ef al., 1993)

histone-2.1.2 Invasion mechanism, replication, and gene expression in host cells of ASFV2.1.2.1 ASFV Invasion Mechanism

ASFV entered host cells a receptor mediated This endocytosis process was

dependent on temperature, energy, cholesterol, and low pH Recent studies had shown

this process to be clathrin-dependent endocytosis (CME) and macro-pinocytosis ASFVinvasion mechanisms included four phases: ASFV adsorption, internalization,uncoating, and release process (Gaudreault e/ al., 2020).

Notably, ASFV primarily infected macrophages There are three routes ASFVtrespassing to the cell: dynamin, clathrin-mediated endocytosis (CME), andmicropinocytosis (Alonso ef al., 2013) This diversity of pathways to enter host cellscould also be found in other viruses (Meier ef a/., 2002)

2.1.2.2 ASFV replication and gene expression mechanism in Host cells

Similar to other large DNA viruses such as poxviruses and herpesviruses, ASFVused a temporal gene expression strategy (Chapman ef al., 2008; Mufioz-Moreno ef al.,2015) There are three phases to the viral RNA polymerase recognizing and starting theexpression throughout the respective stages of the ASFV replication cycle includingearly intermediate and late genes Viral genome replication began in the nucleus andwas subsequently transferred to the cytoplasm, larger intermediates form and mature(Rojo ef al., 1999) DNA replication enzymes were expressed upon entry of the viralcore particles into the cytoplasm (Almendral ef a/., 1990) Virion morphogenesis tookplace in a specific region of the cytoplasm the viral factory (Heath ef a/., 2001)

Around 4 to 6 h post-infection (HPI), early gene expression occurred and

produced necessary proteins for viral replication At 6 to 8 HPI intermediate gene

expression, ASFV replication was started via its DNA polymerase encoded by geneG1211R Intermediate and late gene expression at 8 to 16 HPI produces structuralproteins that were incorporated into the virion

2.1.3 Diseases and associated syndromes

ASFV entered the body via the tonsils or dorsal pharyngeal mucosa and nextmoved to the mandibular or retropharyngeal lymph nodes and the virus spreadsystemically through viremia After that, the virus could be detected in almost all pigtissues The clinical course observed in the ASF of domestic pigs could be described asperacute, acute, subacute, or chronic (Greig, 1972)

2.1.3.1 Peracute ASF

Clinical signs, pathology and manifestation of clinical signs depended ondifferent factors (Schulz ef a/., 2017) Identification sign of the peracute form of ASFVwas characterized by body temperature >41°C, loss of appetite, depression, and

5

cutaneous hyperemia Death swine usually follow 1 to 4 days after clinical signs appear.The peracute form was generally reported in areas without ASFV and the manifestation

of clinical signs were depended on different factors

2.1.3.2 Acute ASF

Acute ASF caused by highly or moderately virulent strains of the virus Its

clinical course characterized by high fever, body temperature of 40 to 42°C, lethargy,

anorexia, inactivity, respiratory failure and severe pulmonary edema, widespreadnecrosis, and hemorrhage such as lymphatic tissue hemorrhage, cutaneous hemorrhage(especially the skin of the ears and flanks), splenomegaly, and high mortality Otherclinical signs such as nasal discharge, vomiting, and diarrhea Diarrhea caused theemergence of black stains in the perianal region of animals (Sanchez-Vizcaino et al.,2015) Pregnant sows may be miscarry (Wu ef al/., 2021)

2.1.3.3 Subacute ASF

Subacute ASF had similar symptoms to the clinical symptoms of acute ASF,although these are generally less pronounced Pigs commonly died 7 to 20 days after

infection with a mortality rate between 30 and 70% Infected pigs exhibited moderate to

high levels of fever The vascular changes, were mostly hemorrhages and edema, in thesubacute form of the disease can be higher than in the acute form (Gomez-Villamandos

et al., 2003) At the post-mortem examination, these pigs had manifestations such as

hydropericardium, ascites, and multifocal edema, very characteristic in the wall of the

gall bladder or the perirenal fat (Sanchez-Vizcaino ef al., 2015) Other clinical signsmight include hemorrhagic splenomegaly or partial splenomegaly, with patches ofspleen affected, and multifocal hemorrhagic lymphadenitis (Gómez-Villamandos ef al.,1995)

2.1.3.4 Chronic ASF

The clinical manifestations of chronic ASF were skin multifocal necrosis,

arthritis, growth retardation, weight loss, dyspnea, and miscarriage

Table 2.1 Main lesions observed in the different forms of ASF (Sanchez ef al., 2015)

Peracute form Acute form Subacute form — Chronic formVirulence High/moderate Moderate Low

x High tan Eien fever, See acute form Respiratory Clinical appetite loss, appetite loss, :

: but less signs,

signs lethargy, lethargy, gastro- tegraynaisl ——

hypernode intestinal signs P

Erythema,

clare petechial Arthritis,

- esin hemorrhages in necrotic skin, Pathology Erythema szszergiÏ ue — several organs, pneumomia,

kumxane cs l hemorrhagic pericarditis,

Làn Wan ° lymph nodes, abortion

abortion

Mortality Variable Low

Figure 2.2 Acute ASF post-mortem examination (4) Severe hemorrhagic

splenomegaly was observed at the opening of the abdominal cavity of an animalwith acute ASF The liver is severely congested (B) A large, dark-coloredspleen with rounded edges (hemorrhagic splenomegaly) occupies a largeabdominal cavity volume in acute ASF (C) Multiple areas of partialhemorrhagic splenomegaly in the spleen from an animal with subacute ASF.(D) Multifocal hemorrhages in a lymph node with a marbled appearance inacute ASF (E) Severe hemorrhagic lymphadenopathy in the gastrohepaticlymph node (arrow) in acute ASF (F) Severe hemorrhagic lymphadenopathy inthe renal lymph node (arrow) in acute ASF (G) Severe hemorrhagiclymphadenopathy in the ileocecal lymph node (arrow) in acute ASF (H)Moderate hemorrhagic lymphadenopathy in the mesenteric lymph node (arrow)

in acute ASF (Salguero, 2020)

pigs and soft ticks (Gallardo ef al., 2018) Several species of soft ticks, such as

O.moubata in Africa and O.erraticus in the Iberian Peninsula are vectors of ASFV(Plowright ef al., 1969; Jori et al., 2023)

ASFV is often transmitted directly via contact between sick and healthy animalsvia oral and nasal routes (Guinat ef a/., 2016) Other routes include tick bite (Plowright

et al., 1969) and injection (intramuscular, subcutaneous, intraperitoneal, or intravenous)(Guinat ef al., 2016) The ASFV incubation period varied from 4 to 19 days, depending

on the ASFV isolate, route of exposure and virulence of the individual strain During

the incubation period, the virus was transmitted from infected domestic pigs, at this timethere are no clinical signs After the onset of clinical symptoms, ASFV had high levels

in all secretions (nasal secretions, saliva, feces, urine, conjunctival secretions, and

genital secretions

ASFV could persist for more than 3 days in contaminated barns and for severalweeks in pig manure At room temperature, ASFV could be isolated after 18 monthsand after 15 weeks in rotting blood (Sanchez-Vizcaino ef al., 2009) In frozen oruncooked meat, ASFV persists for months Incurred or processed products, such asParma ham, infectious virus was not found after 300 days of processing and curing(Fischer et al., 2020) No infectious ASFV was found in cooked or canned hams heated

to 70°C (158°F) Infectious ASFV was undetectable by 110 days in chilled deboned

meat, bone-in meat, or ground pork and by 30 days in smoked deboned meat (Petrini ef

al., 2019)

ASFV was inactivated by organic solvents, detergents, oxidizing agentsincluding hypochlorite and phenol, and commercial disinfectants For example, ASF Vwas inactivated in 30 minutes by exposure to 2.3% chlorine, 3% ortho- phenylphenol,

9

or iodine-containing compounds Other effective virucidal treatments include formalin,

sodium hydroxide, beta-propiolactone, glyceraldehyde’s, or acetyl-ethyleneimine(Sanchez-Vizcainoa ef ạ., 2010)

2.2 Real-time PCR technique

2.2.1 Principles

The real-time PCR method, also known as Quantitative PCR (qPCR), is acombination of the principles of the traditional PCR method and the technique ofdetecting fluorescence signals from amplified products after each reaction cycle.response In the traditional PCR method, after amplifying the target DNA, an additionalstep of electrophoresis on agarose gel must be performed to check the results Real-timePCR when applied to research and diagnosis is often used for qualitative (checkingwhether or not the target gene is present in the sample) and quantitative (determiningthe number of genes present in the sample); Quantification is the outstanding advantage

of Real-time PCR method compared to many other methods, in quantification, there aretwo levels: relative quantification and absolute quantification

The fluorescence is key in the composition of the Real-time PCR reaction so that

the computer can record the signal after each cycle In the presence of the target DNAamplification, this fluorescence caused the tubes to emit fluorescence wavelengths when

receiving excitation light Currently, fluorescence inserted into double-stranded DNA

and probes are the most commonly used fluorescence agents (Monis ef al., 2006)

2.2.2 Reaction components

Real-time fluorescent PCR chemistries are one of the important components of areal-time PCR reaction There are two main proceedings of DNA analysis in real-timePCR: methods enabling both specific and non-specific detection of amplified productsusing dsDNA binding dyes, and those that only detect specific PCR products viaemploying fluorophore-linked oligonucleotides (primer-probes, probes, and analogues

of nucleic acids) (Navarro ef a/., 2015)

2.2.2.1 DNA binding dyes

DNA binding dyes worked based on their ability to absorb excitation light andfluoresce when they were binding with any dsDNA molecules presenting in the reactionmedium This group of luminescent agents included many different chemicals: Ethidium

10

bromide, YO-PRO-1, BEBO, LC Green, SYBR® Green I, SYTO9, SYTO-82 and SYTO-13, EvaGreen, SYBR® Gold.

2.2.2.2 Primer-probes

Primer-probes designed by directly binding elements for fluorescence intoprimers There are many forms of their structure: hairpin (Hairpin primer-probes) ofthree types: scorpion primer-probes, AmplifluorTM primer-probes, and LUXTM (Light-Upon-eXtension) primer-probes; pseudo-cyclic forms called “cyclicons” (Cycliconprimer-probes) or linear structure, co-active with a DNA minor-groove binding dye(Angler® primer-probes) (Navarro ef al., 2015)

2.2.2.3 Probe

The probe is an oligonucleotide sequence with an attached donor and/or acceptorfluorophore, which included two groups, hydrolysis, and hybridization (Navarro et al.,2015) The sequence of nucleotides on the probe designed to be complementary to asequence fragment on the target molecule This has contributed to the increasedspecificity of amplification (Livak ef ai, 1995) One of the probes used is TaqMan,

which has high specificity and applicability (Nagy ef a/., 2017) TaqMan probe belongs

to the group of hydrolysis probes and is a short-chain oligonucleotide, designed tocomplement a region on the target sequence, (Holland ef al., 1991) Normally, the 5'-

end of the probe is associated with 14 a fluorescence emitter (Reporter) and a fluorescent

absorber which is corresponding to the 3-end (Quencher) When receiving the

excitation light, the fluorescence absorber (at the Reporter) changed from the stable state

to the electronically excited state Soon afterward (usually one to ten nanoseconds) theychanged to the intermediate state and began to release energy to return to the initial

stable state (Mergny ef al., 1994) At a certain distance (usually 20 - 80 A) (Lilley and

Wilson, 2000), the energy released from the reporter 1s absorbed by the quencher andemitted in the form of optical (fluorescent quenchers) or heat (dark quenchers) Thanks

to the existence of a reporter and quencher at both ends of the probe, a donor-acceptorFRET (fluorescence resonance energy transfer) pair formed

11

2.2.2.4 Analogues of nucleic acid

primer Fluorescence occurs when

reporter dye and quencher dye are no longer in close proximity

Figure 2 3 Schematic of TaqMan (5’ nuclease) assay (Butler, 2012)

Nucleic acid analogues were compounded that were structurally similar tonaturally occurring nucleic acids, specifically RNA and DNA The most highlightedability of analogues was their stability in biological fluids, their high affinity forcomplementary nucleic acid targets, and their advantages of native DNA (Wang ef al.,2022) In real-time PCR, several analogues of nucleic acids are used such as PeptideNucleic Acids (PNAs), Locked Nucleic Acids (LNAs), Zip Nucleic Acids (ZNAs), andnonnatural bases (iG and 1C) (Navarro ef al., 2015) Besides, other ingredients which

need to used are Thermostable DNA polymerase, DNA template, pair of primers,

deoxynucleotide triphosphates (dNTPs), bivalent cations (Mg?*, Mn”, etc.),

amplification buffer, and deionized water In addition, Uracil - N - glycosylase (UNG)

could be added to prevent external contamination from amplification products of

previous experiments (Green and Sambrook, 2018)

2.2.3 Real amplification chart - PCR heart

In Real-time PCR, the progress and results of the reaction were monitored

through an amplification plot In this diagram, the vertical (Y) axis represented the

fluorescence intensity emitted when receiving the excitation wavelength, and the

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horizontal axis represents the thermal cycles This diagram showed three main stages:the first is the “incubation phase” or the “latent phase” where the sequence of the targetDNA may have been amplified into multiple copies, but the number was still small, notenough for the machine to record the emitted fluorescence signal When reaching acertain stage, the amplification product of the reaction was large enough, the emittedfluorescence signal is large enough for the machine to recognize, the fluorescenceintensity in this stage also increases rapidly after each thermal cycle causes the gain

curve to be pushed high, this stage is called the “exponential phase” Finally, in the

"flattening phase", the fluorescence intensity during this phase begins to slowly increaseand reaches a plateau because free nucleotides have been depleted, the activity ofpolymerase is no longer effective (Tichopad ef a/., 2003)

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of the experimenter The purpose of this method is to know the exact number of copies

of target DNA present in the original specimen to make diagnoses

For accurate quantification of initial copies, a Real-time PCR reaction must beperformed with a standard sample with a known number of copies at different dilutions

to establish a calibration curve on the relationship of the cycles threshold period (Ct)

and number of copies Then, Real-time PCR reaction was performed with test sample to

determine the threshold period (Ct), DNA of the sample was quantified based on therelationship between the standard curve and the threshold cycle (Gibson ef a/., 1996).2.3 Situation of ASFV diagnostic research

2.3.1 National studies

The study performs by Le Thi Hong Hanh in 2019 about detection of African

swine fever virus in Vietnam using real-time PCR This study aimed to detect the

presence of ASFV in Vietnam using real-time PCR The study collected samples ofblood, tissues, and feces from suspected ASF V-infected pigs in different provinces in

Vietnam The results showed that the real-tmme PCR method was able to detect the

presence of ASFV in these samples This result provided important information on theoutbreak of ASFV in Vietnam

"Spatiotemporal dynamics of African Swine Fever virus in Vietnam usingBayesian spatio-temporal models" was researched by Pham Thi Ngoc Tu in 2020 Thisstudy used Bayesian spatio-temporal models to investigate the spatiotemporal dynamics

of ASFV in Vietnam The study analyzed data from ASFV outbreaks in 63 provinces InVietnam from 2018 to 2019 and found that ASFV spread rapidly across the country.The study highlights the importance of early detection and control measures to preventthe spread of ASFV

In 2022, Tran Trong Vuong proclaimed a study to develop a duplex real-timePCR assay for the detection of both ASFV and classical swine fever virus (CSFV) Thestudy collected blood samples from suspected ASF V and CSF V-infected pigs and testedthe samples using the duplex real-time PCR assay The results showed that the assaywas able to detect both ASFV and CSFV with high sensitivity and specificity, thusproviding a useful tool for the diagnosis and surveillance of these two viral diseases 1npigs

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