A simplified PCR test for early detection of dwarf off-types in micropropagated Cavendish bananas (Musa spp. AAA)

The dwarf somaclonal variant is a major problem affecting micropropagation of the banana cultivar Williams (Musa spp. AAA; subgroup Cavendish). This problem arises from genetic changes that occur during the tissue culture process. Early identification of this problem is difficult and propagators must wait until plants are ex vitro in order to visualise the dwarfism phenotype. In this study, we have improved a SCAR-based molecular diagnostic technique, developed by Damasco et al. [Acta Hortic. 461 (1997) 157], for the early identification of dwarf off-types. We have included a positive internal control in a multiplex PCR and adapted the technique for use with small amounts of fresh in vitro leaf material as PCR template. The control product is a 500 bp fragment from 18S rRNA and is amplified in all tissues irrespective of phenotype. The use of small in vitro leaf material removing the need for genomic DNA extraction.

Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR

Aims: The aim of this work was to develop a rapid molecular test for the detection of the Chlamydiaceae family, irrespective of the species or animal host. Methods and Results: The method described herein is a polymerase chain reaction targeting the 16S rRNA gene of the Chlamydiaceae family, and the results demonstrate that the test reacts with five reference Chlamydiaceae but none of the 19 other bacterial species or five uninfected animal tissues tested. The results also indicate the enhanced sensitivity of this test when compared with conventional culture or serology techniques. This is demonstrated through parallel testing of six real clinical veterinary cases and confirmatory DNA sequence analysis. Conclusions, Significance and Impact of the Study: This test can be used by veterinary diagnostic laboratories for rapid detection of Chlamydiaceae in veterinary specimens, with no restriction of chlamydial species or animal host. The test does not differentiate chlamydial species, and if required, speciation must be carried out retrospectively using alternate methods. However, for the purpose of prescribing therapy for chlamydiosis, this test would be an invaluable laboratory tool.

A one-tube fluorescent assay for the quarantine detection and identification of Tilletia indica and other grass bunts in wheat

A molecular assay with enhanced specificity and sensitivity has been developed to assist in the surveillance of Karnal bunt, a quarantineable disease with a significant impact on international trade. The protocol involves the release of DNA from spores, PCR amplification to enrich Tilletia-specific templates from released DNA and a five-plex, real-time PCR assay to detect, identify and distinguish T. indica and other Tilletia species (T. walkeri, T. ehrhartae, T. horrida and a group comprising T. caries, T. laevis, T. contraversa, T. bromi and T. fusca) in wheat grains. This fluorescent molecular tool has a detection sensitivity of one spore and thus bypasses the germination step, which in the current protocol is required for confirmation when only a few spores have been found in grain samples. The assay contains five dual-labelled, species-specific probes and associated species-specific primer pairs in a PCR mix in one tube. The different amplification products are detected simultaneously by five different fluorescence spectra. This specific and sensitive assay with reduced labour and reagent requirements makes it an effective and economically sustainable tool to be used in a Karnal bunt surveillance program. This protocol will also be valuable for the identification of some contaminant Tilletia sp. in wheat grains.

Australian Bat Lyssavirus

In Chapter 1, the literature relating to rabies virus and the rabies like lyssaviruses is reviewed. In Chapter 2, data are presented from 1170 diagnostic submissions for ABLV testing by fluorescent antibody test (Centocor FAT). All 27 non-bat submissions were ABLV-negative. Of 1143 bat accessions 74 (16%) were ABLV-positive, including 69 of 974 (7.1%) flying foxes (Pteropus spp.), 5 of 7 (71.4%) Saccolaimus flaviventris (Yellow-bellied sheathtail bats), none of 151 other microchiropteran bats, and none of 11 unidentified bats. Statistical analysis of data from 868 wild Black, Grey-headed, Little Red and Spectacled flying foxes (Pteropus alecto, P. poliocephalus, P. scapulatus, and P. conspicillatus) indicated that three factors; species, health status and age were associated with significant (p< 0.001) differences in the proportion of ABLV-positive bats. Other factors including sex, whether the bat bit a person or animal, region, year, and season submitted, were not associated with ABLV. Case data for 74 ABLV-positive bats, including the circumstances in which they were found and clinical signs, is presented. In Chapter 3, the aetiological diagnosis was investigated for 100 consecutive flying fox submissions with neurological signs. ABLV (32%), spinal and head injuries (29%), and neuro-angiostrongylosis (18%) accounted for most neurological syndromes in flying foxes. No evidence of lead poisoning was found in unwell (n=16) or healthy flying foxes (n=50). No diagnosis was reached for 16 cases, all of which were negative for ABLV by TaqMan PCR. The molecular diversity of ABLV was examined in Chapter 4 by sequencing 36 bases of the leader sequence, the entire N gene, and start of the P gene of 28 isolates from pteropid bats and 3 isolates from Yellow-bellied sheathtail (YBST) bats. Phylogenetic analysis indicated all ABLV isolates clustered together as a discrete group within the Lyssavirus genera closely related to rabies virus and European bat lyssavirus-2 isolates. The ABLV lineage consisted of two variants; one (ybst-ABLV) consisted of isolates only from YBST bats, the other (pteropid-ABLV) was common to Black, Grey-headed and Little Red flying foxes. No associations were found between the sequences and either the geographical location or year found, or individual flying fox species. In Chapter 5, 15 inocula prepared from the brains or salivary glands of naturally-infected bats were evaluated by intracerebral (IC) and footpad (FP) inoculation of Quackenbush mice in order to select and characterize a highly virulent inoculum for further use in bats (Inoculum 5). In Chapter 6, nine Grey-headed flying foxes were inoculated with 105.2 to 105.5 MICED50 of Inoculum 5 divided into four sites, left footpad, pectoral muscle, temporal muscle and muzzle. Another bat was inoculated with half this dose divided into the footpad and pectoral muscle only. Seven of 10 bats developed clinical disease of 1 to 4 days duration between PI-days 10 and 19 and were shown to be ABL-positive by FAT, HAM immunoperoxidase staining, virus isolation in mice, and TaqMan PCR. Five of the seven bats displayed overt aggression, one died during a seizure, and one showed intractable agitation, pacing, tremors, and ataxia. Viral antigen was demonstrated throughout the central and peripheral nervous systems and in the epithelial cells of the submandibular salivary glands (n=4). All affected bats had mild to moderate non-suppurative meningoencephalitis and severe ganglioneuritis. No ABLV was detected in three bats that remained well until the end of the experiment on day 82. One survivor developed a strong but transient antibody response. In Chapter 7, the relative virulence of inocula prepared from the brains and salivary glands of experimentally infected flying foxes was evaluated in mice by IC and FP inoculation and TaqMan assay. The effects in mice were correlated to the TaqMan CT value and indicated a crude association between virulence and CT value that has potential application in the selection of inocula. In Chapter 8, 36 Black and Grey-headed flying foxes were vaccinated with one (day 0) or two (+ day 28) doses of Nobivac rabies vaccine and co-vaccinated with keyhole limpet haemocyanin (KLH). All bats responded to the Nobivac vaccine with a rabies-RFFIT titer > 0.5 IU/mL that is nominally indicative of protective immunity. Plasma from bats with rabies titres >2 IU/mL had cross-neutralising ABLV titres >1:154. A specifically developed ELISA detected a strong but transient response to KLH.

Rapid multiplex PCR assay for differentiating Pasteurella multocida serovars

To test the robustness and validity of our prototype LPS-specific multiplex PCR on P. multocida field isolates and develop the PCR into a diagnostic test capable of accurately and reliably typing P. multocida strains.

Reliable Means of Diagnosis and Serovar Determination of Blood-Borne Salmonella Strains: Quick PCR Amplification of Unique Genomic Loci by Novel Primer Sets

Typhoid fever is becoming an ever increasing threat in the developing countries. We have improved considerably upon the existing PCR-based diagnosis method by designing primers against a region that is unique to Salmonella enterica subsp. enterica serovar Typhi and Salmonella enterica subsp. enterica serovar Paratyphi A, corresponding to the STY0312 gene in S. Typhi and its homolog SPA2476 in S. Paratyphi A. An additional set of primers amplify another region in S. Typhi CT18 and S. Typhi Ty2 corresponding to the region between genes STY0313 to STY0316 but which is absent in S. Paratyphi A. The possibility of a false-negative result arising due to mutation in hypervariable genes has been reduced by targeting a gene unique to typhoidal Salmonella serovars as a diagnostic marker. The amplified region has been tested for genomic stability by amplifying the region from clinical isolates of patients from various geographical locations in India, thereby showing that this region is potentially stable. These set of primers can also differentiate between S. Typhi CT18, S. Typhi Ty2, and S. Paratyphi A, which have stable deletions in this specific locus. The PCR assay designed in this study has a sensitivity of 95% compared to the Widal test which has a sensitivity of only 63%. As observed, in certain cases, the PCR assay was more sensitive than the blood culture test was, as the PCR-based detection could also detect dead bacteria.

Real-time PCR as a surveillance tool for the detection of Trichinella infection in muscle samples from wildlife

Trichinella nematodes are the causative agent of trichinellosis, a meat-borne zoonosis acquired by consuming undercooked, infected meat. Although most human infections are sourced from the domestic environment, the majority of Trichinella parasites circulate in the natural environment in carnivorous and scavenging wildlife. Surveillance using reliable and accurate diagnostic tools to detect Trichinella parasites in wildlife hosts is necessary to evaluate the prevalence and risk of transmission from wildlife to humans. Real-time PCR assays have previously been developed for the detection of European Trichinella species in commercial pork and wild fox muscle samples. We have expanded on the use of real-time PCR in Trichinella detection by developing an improved extraction method and SYBR green assay that detects all known Trichinella species in muscle samples from a greater variety of wildlife. We simulated low-level Trichinella infections in wild pig, fox, saltwater crocodile, wild cat and a native Australian marsupial using Trichinella pseudospiralis or Trichinella papuae ethanol-fixed larvae. Trichinella-specific primers targeted a conserved region of the small subunit of the ribosomal RNA and were tested for specificity against host and other parasite genomic DNAs. The analytical sensitivity of the assay was at least 100 fg using pure genomic T. pseudospiralis DNA serially diluted in water. The diagnostic sensitivity of the assay was evaluated by spiking log of each host muscle with T. pseudospiralis or T. papuae larvae at representative infections of 1.0, 0.5 and 0.1 larvae per gram, and shown to detect larvae at the lowest infection rate. A field sample evaluation on naturally infected muscle samples of wild pigs and Tasmanian devils showed complete agreement with the EU reference artificial digestion method (k-value = 1.00). Positive amplification of mouse tissue experimentally infected with T. spiralis indicated the assay could also be used on encapsulated species in situ. This real-time PCR assay offers an alternative highly specific and sensitive diagnostic method for use in Trichinella wildlife surveillance and could be adapted to wildlife hosts of any region. (C) 2012 Elsevier B.V. All rights reserved.

Diagnostic Molecular Markers for Phosphine Resistance in U.S. Populations of Tribolium castaneum and Rhyzopertha dominica

Stored product beetles that are resistant to the fumigant pesticide phosphine (hydrogen phosphide) gas have been reported for more than 40 years in many places worldwide. Traditionally, determination of phosphine resistance in stored product beetles is based on a discriminating dose bioassay that can take up to two weeks to evaluate. We developed a diagnostic cleaved amplified polymorphic sequence method, CAPS, to detect individuals with alleles for strong resistance to phosphine in populations of the red flour beetle, Tribolium castaneum, and the lesser grain borer, Rhyzopertha dominica, according to a single nucleotide mutation in the dihydrolipoamide dehydrogenase (DLD) gene. We initially isolated and sequenced the DLD genes from susceptible and strongly resistant populations of both species. The corresponding amino acid sequences were then deduced. A single amino acid mutation in DLD in populations of T.castaneum and R.dominica with strong resistance was identified as P45S in T.castaneum and P49S in R.dominica, both collected from northern Oklahoma, USA. PCR products containing these mutations were digested by the restriction enzymes MboI and BstNI, which revealed presence or absence, respectively of the resistant (R) allele and allowed inference of genotypes with that allele. Seven populations of T.castaneum from Kansas were subjected to discriminating dose bioassays for the weak and strong resistance phenotypes. Application of CAPS to these seven populations confirmed the R allele was in high frequency in the strongly resistant populations, and was absent or at a lower frequency in populations with weak resistance, which suggests that these populations with a low frequency of the R allele have the potential for selection of the strong resistance phenotype. CAPS markers for strong phosphine resistance will help to detect and confirm resistant beetles and can facilitate resistance management actions against a given pest population.

Evaluation of a multiplex PCR to identify and serotypeActinobacillus pleuropneumoniaeserovars 1, 5, 7, 12 and 15

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and Impact of the Study A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease.

Molecular basis for the development of diagnostic methods and specific treatment modalities for idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease with unknown aetiology and poor prognosis. IPF is characterized by alveolar epithelial damage that leads tissue remodelling and ultimately to the loss of normal lung architecture and function. Treatment has been focused on anti-inflammatory therapies, but due to their poor efficacy new therapeutic modalities are being sought. There is a need for early diagnosis and also for differential diagnostic markers for IPF and other interstitial lung diseases. The study utilized patient material obtained from bronchoalveolar lavage (BAL), diagnostic biopsies or lung transplantation. Human pulmonary fibroblast cell cultures were propagated and asbestos-induced pulmonary fibrosis in mice was used as an experimental animal model of IPF. The possible markers for IPF were scanned by immunohistochemistry, RT-PCR, ELISA and western blot. Matrix metalloproteinases (MMPs) are proteolytic enzymes that participate in tissue remodelling. Microarray studies have introduced potential markers that could serve as additional tools for the assessment of IPF and one of the most promising was MMP 7. MMP-7 protein levels were measured in the BAL fluid of patients with idiopathic interstitial lung diseases or idiopathic cough. MMP-7 was however similarly elevated in the BAL fluid of all these disorders and thus cannot be used as a differential diagnostic marker for IPF. Activation of transforming growth factor (TGF)-ß is considered to be a key element in the progression of IPF. Bone morphogenetic proteins (BMP) are negative regulators of intracellular TGF-ß signalling and BMP-4 signalling is in turn negatively regulated by gremlin. Gremlin was found to be highly upregulated in the IPF lungs and IPF fibroblasts. Gremlin was detected in the thickened IPF parenchyma and endothelium of small capillaries, whereas in non-specific interstitial pneumonia it localized predominantly in the alveolar epithelium. Parenchymal gremlin immunoreactivity might indicate IPF-type interstitial pneumonia. Gremlin mRNA levels were higher in patients with end-stage fibrosis suggesting that gremlin might be a marker for more advanced disease. Characterization of the fibroblastic foci in the IPF lungs showed that immunoreactivity to platelet-derived growth factor (PDGF) receptor-α and PDGF receptor-β was elevated in IPF parenchyma, but the fibroblastic foci showed only minor immunoreactivity to the PDGF receptors or the antioxidant peroxiredoxin II. Ki67 positive cells were also observed predominantly outside the fibroblastic foci, suggesting that the fibroblastic foci may not be composed of actively proliferating cells. When inhibition of profibrotic PDGF-signalling by imatinib mesylate was assessed, imatinib mesylate reduced asbestos-induced pulmonary fibrosis in mice as well as human pulmonary fibroblast migration in vitro but it had no effect on the lung inflammation.

The phenanthroimidazole-based dizinc(II) complex as a fluorescent probe for the pyrophosphate ion as generated in polymerase chain reactions and pyrosequencing

A highly selective and sensitive phenanthroimidazole tagged Mannich base type dizinc(II) fluorescent probe (R-Zn2+) has been developed for the pyrophosphate ion (PPi) with a very low limit of detection (LOD) of 0.25 ppm; this also assesses PPi from DNA polymerization chain reaction (PCR).

Design and synthesis of biofunctional magnetic/fluorescent glyco-nanoparticles and quantum dots and their application as specific molecular imaging probes

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Production of positive controls for calcivirus-specific PCR using recombinant baculiovirus technology

Recent advances in our knowledge of the genetic structure of human caliciviruses (HuCVs) and small round-structured viruses (SRSVs) have led to the development of polymerase chain reaction (PCR)-based molecular tests specific for these viruses. These methods have been developed to detect a number of human pathogenic viruses in environmental samples including water, sewage and shellfish. HuCVs and SRSVs are not culturable, and no animal model is currently available. Therefore there is no convenient method of preparing viruses for study or for reagent production. One problem facing those attempting to use PCR-based methods for the detection of HuCVs and SRSVs is the lack of a suitable positive control substrate. This is particularly important when screening complex samples in which the levels of inhibitors present may significantly interfere with amplificiation. Regions within the RNA polymerase regions of two genetically distinct human caliciviruses have been amplified and used to produce recombinant baculoviruses which express RNA corresponding to the calicivirus polymerase. This RNA is being investigated as a positive control substrate for PCR testing, using current diagnostic primer sets. Recombinant baculovirus technology will enable efficient and cost-effective production of large quantities of positive control RNA with a specific known genotype. We consider the development of these systems as essential for successful screening and monitoring applications.

Difterentially expressed genes of Tetrahymena thermophila in response to tributyltin (TBT) identified by suppression subtractive hybridization and real time quantitative PCR

Tributyltin (TBT) is widely used as antifouling paints, agriculture biocides, and plastic stabilizers around the world, resulting in great pollution problem in aquatic environments. However, it has been short of the biomonitor to detect TBT in freshwater. We constructed the suppression subtractive hybridization library of Tetrahymena thermophila exposed to TBT, and screened out 101 Expressed Sequence Tags whose expressions were significantly up- or down-regulated with TBT treatment. From this, a series of genes related to the TBT toxicity were discovered, such as glutathione-S-transferase gene (down-regulated), plasma membrane Ca2+ ATPase isoforms 3 gene (up-regulated) and NgoA (up-regulated). Furthermore, their expressions under different concentrations of TBT treatment (0.5-40 ppb) were detected by real time fluorescent quantitative PCR. The differentially expressed genes of T thermophila in response to TBT were identified, which provide the basic to make Tetrahymena as a sensitive, rapid and convenient TBT biomonitor in freshwater based on rDNA inducible expression system. (c) 2006 Elsevier B.V. All rights reserved.

PCR-RFLP analysis of mitochondrial DNA ND5/6 region among 3 subspecies of common carp (Cyprinus carpio L.) and its application to genetic discrimination of subspecies

Mitochondrial DNA ND5/6 region was studied by PCR-RFLP analysis among ten representative strains belonging to three subspecies (Cyprinus carpio carpio, Cyprinus carpio haematopterus and Cyprinus carpio rubrofuscus) of common carp (Cyprinus carpio L.). A total of 2.4 kb fragment was amplified and subjected to restriction endonuclease analysis with nine restriction endonucleases subsequently. The results indicated that each subspecies owned one hyplotype and four restriction enzymes (Dde I, HaeIII, Taq I and Mbo I) produced diagnostic restriction sites which could be used for discriminating the three subspecies and as molecular genetic markers for assistant selective breeding of common carp.