MINISTRY OF EDUCATION AND TRAININGNONG LAM UNIVERSITY HO CHI MINH CITYFACULTY OF BIOLOGICAL SCIENCESGRADUATION THESIS DETECTION OF Passionfruit Vietnam virus PVNV IN PASSIONFRUIT BY RT-P
Objectives of the research na
Origin of passionfruit na 2.1.2 Botanical features of Passiflora CQUlis ecceccceccesceeceeseeeeeeseeneeeseesenseeeenseeeenseens 3 đố 51C LASSTELCGTTO Tì-:syssoszoyssety0:2062590930853272g8RB)920300210gS02303)28h0992E089y.G0180:8GĐAU2N0E9060023⁄0S0E200138E9 3
Passionfruit (Passiflora edulis), discovered by Spanish explorers in Peru in 1569, symbolizes the passion of Christ, reflecting the explorers' belief in divine approval of their mission Major producers of passionfruit include Brazil, Colombia, Peru, and Ecuador (Deshmukh et al., 2017), with the fruit being introduced to Vietnam in the early 20th century The peel and seeds of passionfruit constitute over 60% of the fruit's weight, making it crucial to utilize these industrial by-products to enhance the supply chain's value Additionally, passionfruit has been widely used in South American folk medicine to treat various ailments, including anxiety, insomnia, asthma, bronchitis, and urinary infections (Viuda-Martos et al., 2020).
2.1.2 Botanical features of Passiflora edulis
The plant features green, angular stems that become round with age and have hollow centers Each stem is adorned with numerous green tendrils and stipules of varying shapes at the base of the leafstalks The leaves are alternate and simple, typically divided into three large, deep lobes, though young plants may have undivided leaves The flowers consist of five alternating sepals and five petals, creating the illusion of ten petals; sepals are green with white interiors, while the thin, white petals have curled edges Surrounding the flower's central hollow are long, thin, curly threads known as the corona, which are violet at the base and white above The central hollow contains a green area with brown, hair-like glands and a small green ovary that eventually matures into fruit (Deshmukh et al., 2017).
The genus Passiflora, commonly known as passionfruit, was first classified by Linnaeus in 1745, identifying 22 species Today, it is estimated that there are over 600 species, many of which are threatened in their natural rainforest habitats The first hybrids were produced in the UK during the 1820s, with P x violacea being one of the notable varieties, although its name is still debated Passiflora is significant within the Passifloraceae family, which is known for its economic importance and widespread distribution in tropical and warm temperate regions (Cerqueira-Silva et al., 2018).
Species: Passiflora edulis Sims 2.1.4 Nutrients of passionfruit
Passionfruit is a nutrient-dense fruit, providing essential vitamins and minerals, particularly fiber, vitamin C, and vitamin A According to the USDA (2020), passionfruit juice consists of 70% to 75% water A single purple passionfruit contains only 17 calories, 4.2 grams of carbohydrates, and 2 grams of fiber Additionally, it offers 6% of the Daily Value (DV) for vitamin C, 5% DV for vitamin A, 2% DV for iron, and 1% DV each for potassium and magnesium.
Passionfruit is a versatile fruit utilized in numerous recipes, ranging from beverages to both savory and sweet dishes Its unique aroma and tangy flavor enhance various culinary creations Frequently, passionfruit juice is combined with ingredients like pineapple, mango, and ginger to elevate the overall taste Additionally, it plays a significant role in confectionery and is commonly used in cakes, pies, and ice cream.
Passionfruit has a diverse range of medicinal uses across various regions In the West Indies, Mexico, South America, and Indonesia, its root is employed as an emetic and diuretic In Brazil, the fruit's pulp serves as a digestive stimulant and tonic, while in Italy, the plant is utilized for its antispasmodic and sedative properties Additionally, in North Eastern India, the leaves are consumed as a vegetable, and a boiled extract of fresh tender leaves is recommended for ailments such as diabetes, hypertension, diarrhea, dysentery, gastritis, abdominal flatulence, and as a liver tonic.
Research by Kawakami et al (2021) reveals that 88% of total polyphenols in passionfruit are concentrated in the seeds, with piceatannol identified as a key stilbene known for its protein-tyrosine kinase inhibition and activation of sirtuin (SIRT), which regulates energy metabolism Additionally, da Silva et al (2013) demonstrated the antioxidant potential of aqueous passionfruit leaf extract, showing a 20% reduction in thiobarbituric acid reactive substances (TBARS) in the liver of treated animals The study also noted a 40% increase in glutathione (GSH) levels in the kidneys, alongside doubled glutathione reductase (GR) activity and a significant decrease in glutathione peroxidase (GPx) activity in the liver Furthermore, animals receiving the extract exhibited a 45% reduction in superoxide dismutase (SOD) activities in both liver and brain These findings suggest that passionfruit leaves could serve as an effective antioxidant supplement to combat oxidative stress.
Wild passionfruit is cultivated in various regions globally, including Java, Sumatra, Malaya, Western Samoa, Norfolk Island, Cook Islands, Solomon Islands, Guam, the Philippines, Ivory Coast, Zimbabwe, and Taiwan The yellow passionfruit, first exported from Brazil to Venezuela in 1954, has since developed into a significant industry and gained popularity in the country.
In 1913, purple passionfruit was brought to Jamaica's Blue Mountains, and today, both purple and yellow varieties are extensively cultivated in regions such as Puerto Rico, Ceylon, Madras, Kerala, and India The global production of passionfruit reaches approximately 1.5 million tonnes, with Brazil dominating the market by contributing around 90% of this total In 2020, the passionfruit industry generated USD 14.5 million from 41,879 metric tons harvested across 3,322 hectares.
Currently, the South Central of Vietnam represents more than 86% of the country's land area and is the primary passionfruit region, spanning over 8,2 thousand hectares.
With a production of more than 134,000 tons and an area of more than 4,263 ha, Gia Lai province is the greatest passionfruit area (Duong, 2023).
Passionfruit woodiness disease, primarily transmitted by aphids, significantly impacts widely cultivated genotypes in Brazil, Peru, Venezuela, South Africa, Sri Lanka, America, and Australia This disease poses a serious threat to passionfruit production, especially during its initial emergence and spread While specialized treatments exist for bacterial, fungal, and nematode infections, preventing passionfruit woodiness disease is crucial to maintaining yields and protecting vulnerable varieties from total loss Cultivating resistant varieties is an effective strategy for controlling this viral disease and ensuring sustainable passionfruit cultivation.
Plant viruses spread through wounds or by vectors, with Mivumoni experiencing the highest disease incidence at 51.43% and Mangawani the lowest at 32.50% Notably, every farm surveyed in Kilifi County showed a 100% prevalence of PWD (Asande et al., 2023).
2.3.2 Cause of Passionfruit Woodiness Disease
The history of Passionfruit Woodiness Disease (PWD) is complex, as multiple viruses can cause similar symptoms First reported in Australia in 1901 by Cobb, the disease's cause remained unidentified for years (Manicom et al., 2003) PWD primarily spreads through aphids, impacting widely cultivated genotypes and leading to significant fruit losses due to the propagation methods of seeds and grafting Managing PWD is challenging due to the non-persistent transmission of various aphid vectors, making effective control difficult (Asande et al., 2023).
The disease is caused by various potyviruses, including the Passionfruit Vietnam Virus, which belongs to the Potyvirus genus within the Potyviridae family This family consists of multiple genera of plant-infecting single-stranded positive-sense RNA viruses characterized by their flexible and filamentous virion particles.
6 the genus Macluravirus with aphid-transmitted members, the genus Jpomovirus with whitefly-transmitted members and the genera Rymovirus and Tritimovirus, with mitetransmitted members (López-Moya et al., 2009).
The Passionfruit Vietnam virus (PVNV), a variant of the Passiflora mottle virus, has been identified in passionfruit crops Its complete genome, measuring 10,029 bp, is available on NCBI GenBank As a newly discovered virus impacting passionfruit, there is limited published research on PVNV Notably, information regarding this virus can be found in the journal of the Department of Plant Pathology at National Chung Hsing University in Taichung City, Taiwan, although it remains unpublished.
PWD is spread by the aphids Myzus persicae and Aphis gossypii through a non-persistent method While these aphids typically do not establish colonies on Passiflora spp., they can transmit the virus during experimental probing Potyviruses, likely including PVNV, are not retained within the aphids and do not remain on their stylets for long durations (Manicom et al., 2003).
ELISA technique 0
The enzyme-linked immunosorbent assay (ELISA) relies on enzymes to indicate the presence and quantity of antigen-antibody interactions There are several types of ELISA, including direct, indirect, sandwich, and competitive assays, each defined by the specific antigen-antibody combination used Enzymes are notably specific and can amplify nonenzymatic reactions significantly, often by up to a billion times Unlike signals from radionuclides or fluorescent compounds, enzyme signals can increase over time as they continuously convert substrates, and with the use of various enzyme-amplification circuits, signal levels can exceed those of radionuclides by two to three orders of magnitude (Butler, 2000).
Polymerase chain reaction (PCR) is a widely used molecular biology technique that enables the enzymatic amplification of DNA without the need for living organisms This method allows for the exponential replication of small DNA samples, facilitating easier analysis PCR is essential in medical and biological research for various applications, including the detection of hereditary diseases, genetic fingerprinting, infectious disease diagnosis, gene cloning, paternity testing, and DNA computing Additionally, PCR can detect viral DNA by using specific primers targeted at viral sequences, making it valuable for diagnostic analyses and viral genome sequencing Its high sensitivity allows for the detection of viruses shortly after infection, often before symptoms arise (Rahman et al., 2013).
RT-PCR is effective with small amounts of RNA, as just one microgram of total RNA, which contains less than 5% mRNA, can generate sufficient cDNA for ten PCR reactions To prevent amplification of contaminating DNA, it is crucial to eliminate any DNA contamination, and designing primers that target regions containing introns can help address this issue.
The reverse transcription reaction design is straightforward, requiring the selection of a primer and reverse transcriptase A DNA primer is essential to initiate the process, with three types available: target specific primers, oligo (dT), and random hexanucleotides Oligo (dT) effectively generates cDNA up to 2-3 kb from polyadenylated RNAs, while random hexanucleotides are ideal for producing cDNA from all RNA types, particularly beneficial for longer RNA sequences exceeding 2-3 kb, which may not be fully reverse transcribed by oligo (dT) Target specific primers, typically the 3'-PCR primers, are less commonly used due to the need for precise concentration optimization in each case (Ohan and Heikkila, 1993).
The Passiflora mottle virus (PaMoV) and Passionfruit Vietnam Virus (PVNV) share identical reference sequence GCF 018583715.1, indicating that they possess similar characteristics Notably, information regarding PVNV has been documented in the journal of the Department of Plant Pathology at National Chung Hsing University, located in Taichung City, Taiwan, but remains unpublished.
Between 2016 and 2017, researchers conducted extensive virus screenings of passion fruit leaves and fruits exhibiting viral symptoms in Fujian Province, China A fruit sample displaying severe mottle symptoms was selected for analysis, and RT-PCR was performed using genus-specific degenerate primers aimed at detecting Potyvirus members The findings revealed an 89% nucleotide and 91% amino acid sequence identity with a previously unreported potyvirus, provisionally named "passionfruit Vietnam potyvirus" (PVNVDakNong) Both PFV-FJ and PVNV-DakNong are classified under the same potyvirus species, with PFV-FJ identified as a new member of the genus Potyvirus, tentatively named "passion fruit severe mottle-associated virus" (Xie et al., 2019).
A field survey from 2017 to 2020 identified significant occurrences of Passion Fruit Woodiness Disease (PWD) in passionfruit plantations across Northern and Southern Vietnam The symptoms observed included severe foliar mosaic, stunted growth, and the production of small, woody, and distorted fruits These findings offer crucial insights into the prevalence of the major Passion Fruit Woodiness Virus (PaMoV) and related pathogens.
In Vietnam, ten potyviruses have been identified as inducing Passionfruit Woodiness Disease (PWD) in passionfruit plants In August 2018, partial genome sequences of four virus isolates were deposited in the NCBI GenBank, identified as Passiflora mottle virus (accession numbers MG087836, MG087835, MG087834, and MG087833) Sequence alignment revealed that these isolates share 86.9% to 96.5% nucleotide and 95% to 97.6% amino acid identities with the DN1 isolate, leading the Potyvirus Study Group to propose this as a new species Field surveys in Vietnam using ELISA and RT-PCR confirmed that both EAPV and PaMoV contribute to PWD, with instances of mixed infections potentially complicating passionfruit production However, simultaneous detection of the viruses was not achieved in all samples due to geographical distribution and limitations in local testing capabilities.
In 2023, Do Duy Hung et al developed a nonpathogenic, attenuated strain of PaMoV for disease control through cross protection They constructed a full-length genomic cDNA of the PaMoV strain DN4 from Vietnam to create an infectious clone In experiments involving 45 plants, the attenuated mutant PaMoV-Ess3lis1 demonstrated a high protection rate of 91% against the wild-type virus in passionfruit plants This study indicates that PaMoV-Es3I1s1 can effectively serve as a protective agent for controlling PaMoV via cross protection.
3.1 Time and location of the research
This graduate thesis had been done on 7/2023 — 10/2023 at A204 — A205 Molecular Biology Room, Research Institute for Biotechnology and Environment (RIBE), Nong Lam University Ho Chi Minh City.
PVNV-infected leaf as positive control and cDNA of ToMV, ToMMV, SPFMV were provided by Research Institute for Biotechnology and Environment.
A total of 34 passionfruit-leaf samples was collected at 3 farms.
Field sample Coodinate Location Amount of field samples
11°97’40.90”N Tan ward, Gia Nghia Farm 3 ; 22 —34
The study utilized a range of specialized equipment, including an Ohaus analytical balance from the USA, a Mupid® One electrophoresis system by Takara Bio from Japan, and a UV light box Additional tools included a Sharp microwave from Japan, a Sanaky refrigerator from Vietnam, and a BioDropTM LITE from the UK The research also employed a Fisher Scientific Mini Centrifuge from the USA, a LifeEco Thermo Cycler from Bioer in China, and a Microflow ABS 1200 biological safety cabinet by Bioquell from the UK.
Tools: sample grinding tools, micropipettes, eppendorf 0.2 mL — 1.5mL — 2mL, pipette tips, cylinders, RNA binding column, test tubes, test tube rack, alcohol burner, petri dishes.
The EZ-10 Spin Column Plant RNA Miniprep Kit (Cat#BS82314) from Biobasic, Canada, is essential for RNA extraction, while the DNase I, RNase-free Kit (Lot1003634) from Thermo Scientific, USA, ensures the integrity of RNA samples For cDNA synthesis, the SensiFASTTM cDNA Synthesis Kit (Cat#BIO-65054) by Meridian Life Science is highly effective, complemented by MyTaqTM Mix (Cat#BIO-25041) for robust PCR performance Additionally, the CloneJET PCR Cloning Kit (Cat#K-) facilitates efficient cloning processes, making these tools vital for molecular biology applications.
1232, Thermo Scientific, USA), Agarose (Cat#BIO-41025, Meridian Life Science), primer (Phu Sa Genomics), GelRedTM loading buffer with Tricolor (TBR, Vietnam),
HyperLadder TM 100 bp (Cat#BIO-33056, Bioline, Anh), TAE 10X (Cat#CV-43, BioBasic, Canada), GeneJET Plasmid Miniprep Kit (Cat#K-0502, Thermo Scientific, USA).
[ RNA extration by EZ-10 Spin Column Plant RNA Miniprep Kit
Perform RT-PCR, electrophoresis and sequencing
[ Cloning target gene by CloneJET PCR Cloning Kit
Optimize annealing temperature and concentration of primer Determine the specificity and sensitivity (LOD) of RT-PCR test
[ Application of the established RT-PCR assay on field samples
Figure 3.1 Workflow of this study.
PCR primers are short, single-stranded segments of DNA that are designed to be complementary to the beginning and end of the target sequence that will be amplified.
In a PCR, it is the primers that dictate exactly what sequence of DNA gets copied.
In primer design, several critical factors must be taken into account, including primer length, the length difference between primer pairs, GC content, nucleotide composition at the 3’ end, PCR product size, and the concentrations of PCR buffer reagents Additionally, stable secondary structures and the melting temperature (Tm) of the primers are essential, with successful PCR typically requiring Tm values between 50°C and 62°C It is crucial that the melting temperature difference between primer pairs does not exceed 5°C to ensure effective PCR performance.
Primers design based on the published conserved sequence genome of PVNV from NCBI Genbank Using NCBI “Primer BLAST” is a common tool to check for non — specific binding.
RNA extraction was performed using the EZ-10 Spin Column Plant RNA Miniprep Kit (Biobasic) following a specific protocol Initially, 450 µL of Buffer Rlysis-PG was added to RNase-free 1.5 mL centrifuge tubes, and 20-50 mg of plant tissue was ground in the same tube The mixture was inverted to ensure thorough mixing and incubated at room temperature for 5 minutes to achieve complete cell lysis After centrifugation at 12,000 x g for 5 minutes, the supernatant was transferred to a new RNase-free tube An equal volume of ethanol was added, mixed by inversion, and the solution was transferred to a spin column, followed by centrifugation at 12,000 x g for 30 seconds The flowthrough was discarded, and 0.5 mL of Universal GT Solution was added, centrifuged, and the flowthrough discarded again This process was repeated with 0.5 mL of Universal NT Solution A final centrifugation step at 12,000 x g for 30 seconds ensured complete removal of residual ethanol Finally, the column was placed in a new 1.5 mL centrifuge tube, 50 µL of RNase-free water was added, and the solution was kept at room temperature for further use.
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The experiment utilized DNase I and an RNase-free Kit from Thermo Scientific, USA, to maintain RNA integrity All reagents and materials were prepared on ice within a Microflow ABS 1200 biological safety cabinet to ensure optimal RNA quality.
Table 3.2 Reagent to remove DNA contamination
Component Stock Final amount Volume (nL) RNA Extraction - - 8
The mixture was incubated in the thermacycler at 37°C for 30 minutes, then the reaction was inactivated by adding 1 pL EDTA and incubated at 65°C for 10 minutes. Stored the products at -20°C.
3.4.6 cDNA synthesis by Reverse Transcription cDNA synthesis is a process of producing complementary DNA (cDNA) by reverse transcription The process utilizes mRNA or miRNA as a template, reverse transcriptase enzyme, and a thermostable primer, complementary to the 3’ end of the RNA template.
In the experiment, all reagents and materials were prepared on ice within a biological safety cabinet (Microflow ABS 1200) The thermal cycling conditions for the reaction included a sequence of temperatures: 25°C for 10 minutes, followed by 42°C for 15 minutes, then 48°C for 15 minutes, and concluding with 85°C for 5 minutes, before holding the final mixture at 4°C.
Component Stock Final amount Volume (uL) TransAmp Buffer 5X 1X 4
3.4.7 Polymerase chain reaction and electrophoresis
Polymerase Chain Reaction (PCR) is an essential molecular biology technique that enables the amplification of DNA, allowing researchers to generate millions of copies of a specific DNA sequence from minimal starting material Each test included negative controls to ensure accuracy The amplification process utilized specific reagents (refer to table 3.4) and followed a designated thermocycle program (see table 3.5) to effectively detect PVNV.
Electrophoresis is the process by which electrically charged molecules migrate in an electric field, allowing for the separation of proteins or DNA in a gel matrix This technique is widely utilized in life sciences, employing various methods tailored to the size and type of molecules In molecular biology, TAE buffer is commonly used in agarose electrophoresis for nucleic acid separation, including DNA and RNA For instance, RT-PCR products can be analyzed using a 1.5% agarose gel at a voltage of 100V for 30 minutes, followed by visualization under UV light.
Component Stock Final amount Volume (nL) Master mix 2X 1X 6.25
Reverse primer 10 uM 0.2 uM 0.25 cDNA - - 1
3.4.8 Sequencing of the obtained PCR products
Sanger sequencing, or the chain termination method, is a DNA sequencing technique that relies on the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication Developed by Frederick Sanger and colleagues in 1977, it was the predominant sequencing method for about 25 years before being supplanted by next-generation sequencing (NGS) technologies.
The PCR products were sequenced using both forward and reverse primers through the Sanger sequencing method at the Institute of Molecular Biology Loci To establish genetic identity, the sequencing results were analyzed with the BLAST tool, which compared regions of similarity between the obtained sequences and those available in the NCBI Genbank.
Using a cloning vector and restriction enzymes, a novel recombinant DNA molecule is created by inserting a DNA fragment of interest This recombinant DNA is introduced into bacterial cells via a transformation process, enabling the bacteria to replicate the recombinant DNA After the transformation, the bacteria are allowed to rest and then plated on agar plates for growth and propagation.
The CloneJET PCR Cloning Kit (Thermo Scientific) was utilized to clone the target gene, with transformed bacteria cultured overnight on Luria-Bertani agar media supplemented with 100 mg/L ampicillin at 37°C Plasmids containing the viral gene were extracted using the GeneJET Plasmid Miniprep Kit Verification of the cloning vector's efficiency and sequencing of the cloned insert were performed using the pJET1.2 Forward and Reverse Sequencing Primers provided in the kit Colony PCR and sequencing confirmed the successful cloning of the target gene in the bacterial colonies.
17 recombinant plasmids were chosen for further works This cloning vector plasmid used as positive control for all experiments.
Improper annealing temperatures can lead to issues in PCR, where a low annealing temperature (Ta) results in the amplification of non-specific DNA fragments, producing multiple bands on agarose gels Conversely, a high Ta can decrease the yield and purity of the desired product due to ineffective primer annealing To optimize primer functionality, a temperature range of 56°C to 65°C was determined based on the theoretical melting temperatures calculated using NCBI Primer BLAST Detailed information on the reagents and thermocycle program is provided in tables 3.6 and 3.7.
The PCR products were analyzed by 1.5% agarose gel with the voltage of 100V in
For optimal results in RT-PCR reactions, a 30-minute annealing period followed by visualization under UV light is essential, as indicated by the clarity and brightness of the product band Table 3.6 outlines the reagents necessary for optimizing the PCR annealing temperature.
Component Stock Final amount Volume (pL) Master mix 2X 1X 6.25
Reverse primer 10 uM 0.2 uM 0.25 cDNA - - 1
Table 3.7 Thermocycle program for PCR annealing temperature optimization
Primers were tested at various concentrations of 0.025 uM, 0.05 uM, 0.1 uM, 0.2 uM, 0.3 uM, and 0.4 uM, as detailed in Tables 3.8 and 3.9, along with the thermocycle program It was observed that higher primer concentrations could lead to excessive unused primers, potentially causing non-specific amplification products, while lower concentrations might limit the reaction.
The PCR products were analyzed by 1.5% agarose gel with the voltage of 100V in
For optimal results in RT-PCR reactions, a 30-minute incubation followed by visualization under UV light is essential The ideal concentration of primers is indicated by the brightness and clarity of the product band Refer to Table 3.8 for the reagents used in optimizing PCR primer concentrations.
Component Stock Final amount Volume (pL) Master mix 2X 1X 6.25
Forward primer 1.25uM—20uM 0.025 uM—0.4 uM 0.25
Reverse primer 1.25nuM-—20uM ~~ 0.025 nM—0.4 uM 0.25 cDNA - - 1
Table 3.9 Thermocycle program for PCR primers concentration optimization
3.4.12 Evaluation of the specificity of the RT-PCR
The specificity of the RT-PCR assay was evaluated by testing ToMV, ToMMV, and SPFMV using the PVNV primer Optimization of the annealing temperature and primer concentration was conducted to enhance the accuracy of the PCR results, as detailed in sections 3.4.10 and 3.4.11.
The PCR products were analyzed by 1.5% agarose gel with the voltage of 100V in
30 minutes followed by visualization under UV light The proper specificity for a RT- PCR reaction was indicated by the brighter and clearer of the product band.
Table 3.10 Information on the viruses used to detemine specificity of RT-PCR
Abbreviation Virus Host plant Symptom
ToMV virus Solanaceous Irregular, off-color spots family fruit.
Tomato mottle Leaf deformation, ToMMV - ; mosaic virus mosaic, and necrosis.
Sweet potato Convolvulaceae degree of stress, growth
SPFMV feathery mottle virus ; ; family stage, and strain virulence.
3.4.13 Sensitivity and determination for limit of detection (LOD) of the RT-PCR
The sensitivity of RT-PCR was optimized by adjusting the annealing temperature and primer concentrations as detailed in sections 3.4.10 and 3.4.11 Plasmid concentration (ng/µL) was measured using a BioDrop HLITE spectrophotometer, with DNA plasmid samples diluted to various concentrations.
10° to 10° after being calculated by using formula The formula was used to calculate the number of gene copies (Staroscik, 2004):
Copies Where PC: Amount of plasmid concentration (ng)
Application of the established RT-PCR assay on field samples
A total of 34 field samples were randomly collected from three farms in Thu Duc City, Dak Lak, and Dak Nong, focusing on various aspects such as morphology and disease symptoms RNA was extracted from the leaves, and its concentration and purity were measured through absorbance Genomic DNA was eliminated, allowing for the transcription of pure RNA into cDNA The samples were then analyzed using an optimized RT-PCR procedure, and the presence of the virus was confirmed through electrophoresis results.
RESULTS AND DISCUSSION 0.0 cceeeecssesseessesessessesenesneneneaeneenees 22 4.1 Results of establishment of RT-PCR assay for the detection of PVNV by using
Sensitivity and determination for limit of detection (LOD) of the RT-PCR
4.1.9 Sensitivity and determination for limit of detection (LOD) of the RT- PCR
The plasmid concentration was measured using a BioDrop wLITE spectrophotometer at 260 nm Subsequently, a 10-fold gradient dilution of the plasmid samples was performed to assess the limit of detection (LOD) of the established RT-PCR assay The copy number from the plasmid concentration was calculated and diluted from 10^0 to 10^10 to evaluate the sensitivity and LOD of the RT-PCR assay.
The electrophoresis results for the limit of detection (LOD) of the RT-PCR assay are illustrated in Figure 4.8, featuring a DNA 100 bp ladder, a positive control, and a negative control Dilutions ranging from 10° to 10° are represented as samples (1) to (10) The red frame highlights the sensitivity of the RT-PCR assay, demonstrating its effectiveness in detecting target DNA.
Table 4.4 Repetitions of the RT-PCR for determining the limit of detection (LOD) Concentration Run
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Application of the established RT-PCR assay on field samples
Table 4.5 Results of established RT-PCR assay on field samples
Field samples Presence of symtomp RT-PCR result
The optimized RT-PCR assay was employed to detect the presence of PVNV in passionfruit leaf samples from the field, with RNA quality and yield detailed in Table 1 of Appendix 2, confirming suitability for the experiment Out of 34 samples tested, 9 (26.47%) were positive for the PVNV gene Notably, all samples exhibiting symptoms of Passion Fruit Woodiness Disease (PWD) were negative for PVNV, indicating that despite the presence of viral symptoms, the virulent PVNV gene was not detected.
This study aimed to detect the PVNV virulence gene in passionfruit, utilizing RT-PCR on field samples Out of 34 samples tested, 9 (26.47%) were positive for the PVNV gene While samples from Thu Due City showed viral infection, those from Dak Lak and Dak Nong, key passionfruit regions, tested negative The negative results may be attributed to the specific type of passionfruit plant used, as a virus-free TN-1 seedling variety was introduced and cultivated in farms, ensuring initial safety for propagation Nonetheless, it is essential for farmers to implement measures to prevent and control viral vectors in passionfruit crops post-planting.
Passionfruit production in Vietnam and Southeast Asia faces significant challenges due to PWD, with limited encouragement for transgenic virus resistance The necessity for disease control restricts cultivation areas, limits crop diversity in contaminated fields, and requires extensive use of agrochemicals for seed treatment, soil fumigation, plant spraying, and postharvest fruit treatments These control measures not only increase food production costs but also pose risks to human health and the environment due to the harmful effects of toxic chemicals.
The tropical monsoon climate fosters ideal conditions for insects, posing significant risks to passion fruit cultivation To mitigate these threats, it is crucial for farmers and governments to implement effective prevention and control measures, especially in light of emerging viruses The swift proliferation of disease-causing viruses complicates management efforts for passion fruit and other crops Additionally, many virus-infected plants remain asymptomatic in their early stages, making early detection and intervention challenging.
Identifying plant viruses promptly is essential for effective management and control While there are various resources available for viral sequences, most are tailored to specific virus families, particularly those linked to significant infectious diseases like PVNV and PaMoV.
RT-PCR is an effective strategy for early detection of viruses, crucial for controlling passionfruit woodiness disease The method developed in this study offers a reliable tool for rapid screening, helping to manage virus spread and prevent epidemic outbreaks This optimized two-step RT-PCR assay is the first report for detecting PVNV and provides a convenient, sensitive, and cost-effective solution for identifying the virus in diseased fruit plants Additionally, it is designed to be less time-consuming, making it ideal for large-scale surveys, ultimately enhancing productivity and crop quality while reducing expenses.
CONCLUSION AND RECOMMENDATTION
A robust RT-PCR assay was developed to detect the presence of Passionfruit Virus N (PVNV) in passionfruit leaves, achieving optimal results at an annealing temperature of 59°C and a primer concentration of 0.2 µM This assay demonstrated specificity for PVNV, with a detection limit of 10 copies of the target DNA plasmid When applied to field samples, the assay identified PVNV in 9 out of 34 samples, indicating a prevalence rate of 26.47% among the tested plants.
The optimal RT-PCR assay should be implemented on a wider range of field samples for further data about the accuracy of the assay.
To enhance the specificity of RT-PCR assays, it is essential to carefully evaluate the primer pair used Additionally, implementing a two-step RT-PCR process in conjunction with a one-step RT-PCR can help prevent contamination.
1 Hai Dương (2023) Phát triển cây chanh leo bền vững, mang lại thu nhập 6n định cho nông dân https://nhandan.vn/phat-trien-cay-chanh-leo-ben-vung- post767581.html
2 Nguyễn Thị Hoàng Yến (2009) Chanh dây và ứng dụng Nhà xuất bản Hà Nội.
A recent study by Asande et al (2023) published in BMC Plant Biology investigates the prevalence of passion fruit woodiness disease in the coastal lowlands of Kenya The research also evaluates various passion fruit genotypes for their resistance to this disease, highlighting the importance of identifying resilient cultivars to enhance agricultural sustainability in the region.
4 Baker, C A., Jeyaprakash, A., Webster, C G., & Adkins, S (2014) Viruses infecting passiflora species in Florida Florida Department of Agriculture and Consumer Services Tallahassee, FL, USA, 415.
5 Butler, J E (2000) Enzyme-linked immunosorbent assay Journal of immunoassay, 21(2-3), 165-209.
Cerqueira-Silva et al (2014) explore the history of passion fruit woodiness disease, highlighting its impact on Brazilian agriculture The article discusses the current state of this disease in Brazil and its implications for passion fruit cultivation The authors emphasize the need for effective management strategies to combat this disease and ensure the sustainability of the passion fruit industry in the region.
7 Cerqueira-Silva, C B M., Faleiro, F G., de Jesus, O N., dos Santos, E S L., & de Souza, A P (2018) Passion fruit (Passiflora spp.) breeding Advances in Plant Breeding Strategies: Fruits: Volume 3, 929-951.
8 Chen, B., Wu, D., Zheng, H., Li, G., Cao, Y., Chen, J., Yan, F., Song, X., & Lin,
L (2021) Complete genome sequence of passiflora virus Y infecting passion fruit in China Archives of virology, 166, 1489-1493.
9 Chuang, L.-Y., Cheng, Y.-H., & Yang, C.-H (2013) Specific primer design for the polymerase chain reaction Biotechnology letters, 35, 1541-1549.
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M., Paschoal, J A R., Bogusz Junior, S., Furlan, M F., Reyes, F G R., Augusto, F.,
Maróstica Junior, M R., & de Lima Zollner, R (2013,) Antioxidant activity of aqueous extract of passion fruit (Passiflora edulis) leaves: In vitro and in vivo study. Food Research International, 53(2), 882-890.
11 Deshmukh, N A., Patel, R K., Okram, S., Rymbai, H., Roy, S S., & Jha, A K.
(2017) Passion fruit (Passiflora spp.) Magnesium (mg/litre), 100, 200.
12 Do, D H., Chong, Y H., Ha, V C., Cheng, H W., Chen, Y K., Bui, T N L., & Yeh, S D (2021) Characterization and detection of Passiflora mottle virus and two other potyvirus causing passionfruit woodiness disease in Vietnam Phytopathology, 111(9), 1675-1685.
13.Do, D H., Nguyen, T B N., Ha, V C., Raja, J A., & Yeh, S D (2023). Generation of Attenuated Passiflora Mottle Virus Through Modification of the Helper Component Protease for Cross Protection Phytopathology, 113(8), 1605- 1614.
A study by Garcéz et al (2015) investigated the aphid population in yellow passion fruit crops and its correlation with the spread of the Cowpea aphid-borne mosaic virus in a subtropical region of Brazil The research highlights the impact of aphids on agricultural health and disease transmission, emphasizing the need for effective pest management strategies to mitigate viral outbreaks in crops.
15 Joy, J (2022) Importance and value added products of Passiflora edulis The Pharma Innovation Journal, 11(7), 1513-1518.
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19 Lépez-Moya, J J., Valli, A., & Garcia, J A (2009) Potyviridae eLS, 1-10.
20 Savary, S., Willocquet, L., Pethybridge, S J., Esker, P., McRoberts, N., & Nelson,
A (2019) The global burden of pathogens and pests on major food crops Nature ecology & evolution, 3(3), 430-439.
21.Manicom, B., Ruggiero, C., Ploetz, R C., & Goes, A d (2003) Diseases of passion fruit Diseases of tropical fruit crops, 413-441.
22.Marone, M., Mozzetti, S., De Ritis, D., Pierelli, L., & Scambia, G (2001). Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample Biological procedures online, 3, 19-25.
23 Ochwo-Ssemakula, M., Sengooba, T., Hakiza, J., Adipala, E., Edema, R., Redinbaugh, M., Aritua, V., & Winter, S (2012) Characterization and distribution of a Potyvirus associated with passion fruit woodiness disease in Uganda Plant Disease, 96(5), 659-665.
24 Ohan, N W., & Heikkila, J J (1993) Reverse transcription-polymerase chain reaction: An overview of the technique and its applications Biotechnology advances, 11(1), 13-29.
25.Rahman, M T., Uddin, M S., Sultana, R., Moue, A., & Setu, M (2013). Polymerase chain reaction (PCR): a short review Anwer Khan Modern Medical College Journal, 4(1), 30-36.
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28 Staroscik, A (2004) Calculator for determining the number of copies of a template URI Genomics & Sequencing Center, 19, 2012.
29.USDA (2020) Passionfruit, raw United States Department of Agriculture. https://fde.nal.usda.gov/fdcapp.html#/fooddetails/1102676/nutrients
30 Viuda-Martos, M., Pérez-Alvarez, J A., & Fernandez-Lopez, J (2020) Chapter
36 - Passion fruit In A K Jaiswal (Ed.), Nutritional Composition and Antioxidant Properties of Fruits and Vegetables (pp 581-594) Academic Press.
31 Xie, L., Gao, F., Zheng, S., Zhang, X., Zhang, L., & Li, T (2019) Molecular characterization of a new potyvirus infecting passion fruit Archives of virology, 164, 1903-1906.
100 bp — —— VỊ > AP HA seo am nu -
Note: (L) DNA 100 bp Ladder; (+) positive control;
Table 1 RNA quantification of field samples
Table 1 (continue) RNA quantification of field samples
Table 2 Results of established RT-PCR assay on field samples
Field samples Presence of symtomp RT-PCR result