Development and optimisation of a qPCR assay for the enumeration of Cryptophlebia leucotreta granulovirus (CrleGV) used for commercial applications
- Authors: Mela, Thuthula
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362949 , vital:65377
- Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
- Authors: Mela, Thuthula
- Date: 2022-10-14
- Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/362949 , vital:65377
- Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
- Full Text:
- Date Issued: 2022-10-14
The diversity of root fungi associated with Erica species occurring in the Albany Centre of Endemism
- Authors: Bizabani, Christine
- Date: 2015
- Subjects: Ericaceae , Ericas , Roots (Botany) -- Diseases and pests , Mycorrhizal fungi , Polymerase chain reaction , Fungi -- Classification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4160 , http://hdl.handle.net/10962/d1018575
- Description: South Africa has the highest species diversity of ericaceous plants belonging to the Erica genus. There are over 850 identified species in the Cape Floral Region. The Albany Centre of Endemism (ACOE) is located within this region and is a hotspot of diversity consisting of various plant genera. The success of Erica plants is ubiquitously attributed to mycorrhizal relationships they engage in with a diverse group of fungi. This symbiosis is known as the ericoid mycorrhizal (ERM) association. The overall aim of this study was to establish the diversity of root fungi associated with Erica plants using morphological, molecular and 454 pyrosequencing techniques. Six Erica species were identified using leaf and flower morphology according to taxonomic keys. The identified plants were Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Roots from sampled plants were stained and examined microscopically to determine their mycorrhizal status. Ericoid mycorrhizal associations together with dark septate endophyte (DSE) structures and hyphae that did not form any specific structure were observed in all the roots. In addition arbuscular mycorrhizal (AM) structures in the form of vesicles were detected in E. glumiflora and E. cerinthoides. In order to identify the culturable fungi associated with the respective hosts, sterilised roots were placed on various culture media for cultivation. Thereafter isolated fungi were morphologically classified into 67 morphotypes. These were mostly sterile and darkly pigmented. Non-sporulating mycelia of variable colouration such as white, cream-yellowish, beige, green and brown were also observed. Further identification was carried out using molecular techniques. DNA was extracted separately from pure cultures and amplified using ITS1 and ITS4 primers in a polymerase chain reaction (PCR). Thereafter sequencing and Basic Local Alignment Search Tool (BLAST) were used to identify the isolates to generic level. The fungi were taxonomically classified into 54 operational taxonomic units and 94 percent were Ascomycetes and Helotiales was the dominant order. Unclassified Helotiales with affinities to fungi currently identified as Epacrid root fungus was common in all hosts. Other isolates that were identified included Oidiodendron, Meliniomyces, Phialocephala, Cadophora, Lachnum, Leohumicola Cryptosporiopsis, Chaetomium, Acremonium and Epicoccum species. Basidiomycetes were represented by two OTUs belonging to the genus Mycena. Four OTUs comprised fungi that had no significant alignments in the reference databases. Direct root DNA extraction together with 454 pyrosequencing was used to detect the diversity of culturable and unculturable fungi associated with the identified hosts. The ITS2 region was targeted for sequencing. Although Ascomycetes remained the dominant phyla, Basidiomycetes were also detected in all host plants. Glomeromycota was present in E. caffra and E. cerinthoides. Helotiales was dominant in all Erica plants with the exception of E. cerinthoides and E. chamissonis which were dominated by the order Chaetothyriales. The OTUs identified to genus level included Epacris pulchella root fungus, Oidiodendron cf. maius, Acremonium implicatum, Leohumicola, Lachnum, Capronia and Mycena species. Culture-based techniques and pyrosequencing detected similar fungal composition comprising Ascomycetes, while, pyrosequencing was able to detect Glomeromycetes and Basidiomycetes.
- Full Text:
- Date Issued: 2015
The diversity of root fungi associated with Erica species occurring in the Albany Centre of Endemism
- Authors: Bizabani, Christine
- Date: 2015
- Subjects: Ericaceae , Ericas , Roots (Botany) -- Diseases and pests , Mycorrhizal fungi , Polymerase chain reaction , Fungi -- Classification
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4160 , http://hdl.handle.net/10962/d1018575
- Description: South Africa has the highest species diversity of ericaceous plants belonging to the Erica genus. There are over 850 identified species in the Cape Floral Region. The Albany Centre of Endemism (ACOE) is located within this region and is a hotspot of diversity consisting of various plant genera. The success of Erica plants is ubiquitously attributed to mycorrhizal relationships they engage in with a diverse group of fungi. This symbiosis is known as the ericoid mycorrhizal (ERM) association. The overall aim of this study was to establish the diversity of root fungi associated with Erica plants using morphological, molecular and 454 pyrosequencing techniques. Six Erica species were identified using leaf and flower morphology according to taxonomic keys. The identified plants were Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Roots from sampled plants were stained and examined microscopically to determine their mycorrhizal status. Ericoid mycorrhizal associations together with dark septate endophyte (DSE) structures and hyphae that did not form any specific structure were observed in all the roots. In addition arbuscular mycorrhizal (AM) structures in the form of vesicles were detected in E. glumiflora and E. cerinthoides. In order to identify the culturable fungi associated with the respective hosts, sterilised roots were placed on various culture media for cultivation. Thereafter isolated fungi were morphologically classified into 67 morphotypes. These were mostly sterile and darkly pigmented. Non-sporulating mycelia of variable colouration such as white, cream-yellowish, beige, green and brown were also observed. Further identification was carried out using molecular techniques. DNA was extracted separately from pure cultures and amplified using ITS1 and ITS4 primers in a polymerase chain reaction (PCR). Thereafter sequencing and Basic Local Alignment Search Tool (BLAST) were used to identify the isolates to generic level. The fungi were taxonomically classified into 54 operational taxonomic units and 94 percent were Ascomycetes and Helotiales was the dominant order. Unclassified Helotiales with affinities to fungi currently identified as Epacrid root fungus was common in all hosts. Other isolates that were identified included Oidiodendron, Meliniomyces, Phialocephala, Cadophora, Lachnum, Leohumicola Cryptosporiopsis, Chaetomium, Acremonium and Epicoccum species. Basidiomycetes were represented by two OTUs belonging to the genus Mycena. Four OTUs comprised fungi that had no significant alignments in the reference databases. Direct root DNA extraction together with 454 pyrosequencing was used to detect the diversity of culturable and unculturable fungi associated with the identified hosts. The ITS2 region was targeted for sequencing. Although Ascomycetes remained the dominant phyla, Basidiomycetes were also detected in all host plants. Glomeromycota was present in E. caffra and E. cerinthoides. Helotiales was dominant in all Erica plants with the exception of E. cerinthoides and E. chamissonis which were dominated by the order Chaetothyriales. The OTUs identified to genus level included Epacris pulchella root fungus, Oidiodendron cf. maius, Acremonium implicatum, Leohumicola, Lachnum, Capronia and Mycena species. Culture-based techniques and pyrosequencing detected similar fungal composition comprising Ascomycetes, while, pyrosequencing was able to detect Glomeromycetes and Basidiomycetes.
- Full Text:
- Date Issued: 2015
Isolation and identification of Beta-Lactam Producing Microorganisms using PCR based methodologies
- Authors: Krallis, Myrsini
- Date: 1997
- Subjects: Polymerase chain reaction , Bacterial genetics , Fungi -- Genetics , Beta lactam antibiotics , Microbial enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4155 , http://hdl.handle.net/10962/d1018237
- Description: The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.
- Full Text:
- Date Issued: 1997
- Authors: Krallis, Myrsini
- Date: 1997
- Subjects: Polymerase chain reaction , Bacterial genetics , Fungi -- Genetics , Beta lactam antibiotics , Microbial enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4155 , http://hdl.handle.net/10962/d1018237
- Description: The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.
- Full Text:
- Date Issued: 1997
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