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WO1998053074A1 - Adn ribosomique a sous-unites de grande taille, de l'espece $i(neospora) - Google Patents

Adn ribosomique a sous-unites de grande taille, de l'espece $i(neospora) Download PDF

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WO1998053074A1
WO1998053074A1 PCT/AU1998/000367 AU9800367W WO9853074A1 WO 1998053074 A1 WO1998053074 A1 WO 1998053074A1 AU 9800367 W AU9800367 W AU 9800367W WO 9853074 A1 WO9853074 A1 WO 9853074A1
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pcr
sequence
neospora
lsu
dna
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John Timothy Ellis
Cheryl Louise Ryce
Georgina Grace Amoyal
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Insearch Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the present invention is directed to the large subunit (LSU) ribosomal DNA of Neospora species and uses thereof.
  • LSU large subunit
  • the latest release of the LSU ribosomal database contains 429 sequences, yet only 10 (6 nuclear and 4 mitochondrial) are derived from parasites of the phylum Apicomplexa. Three of these nuclear gene sequences (all Toxoplasma gondii) were previously contained in the 1994 release of the database.
  • LSU rRNAs of T. gondii and Plasmodium falciparum have been discussed in some detail and are available from the rRNA WWW Server at URL http://rrna.uia.ac.be/lsu/.
  • These rRNAs conform to the universally accepted core secondary structure proposed for the LSU rRNA which consists of a central multi-branched loop from which helices emanate.
  • the structures branching from the loop are labelled A to I, and within these branches the helices are numbered from 5' to 3 1 .
  • Within the core structure are scattered variable domains known as expansion segments. The origin of these segments is unknown but they always occur at the same place in the LSU structure although they vary in size between taxa. In T.
  • Neospora caninum is a cyst-forming coccidian parasite which is recognised as being closely related to T. gondii. It causes neuromuscular disease in dogs and is now also recognised as a significant cause of abortion and neonatal mortality in livestock such as cows and goats.
  • the genome organisation of Neospora has not been extensively investigated, although current evidence from rDNA comparisons show Neospora and Toxoplasma to be genetically very similar.
  • cPCR Competitive PCR
  • an internal reference standard also frequently called the competitor sequence or PCR MIMIC
  • the PCR MIMIC is typically a synthetic molecule that may be made in a wide variety of ways.
  • Competitive PCR has found a practical use in the quantification of a number of infectious micro-organisms in biological specimens.
  • PCR may be used in one of two ways in order to determine the amount of target sequence in a biological specimen.
  • the PM may be titrated in the presence of a constant amount of target sequence and the equivalence point determined where the PM and the target generate PCR products of similar yield.
  • serial dilutions of target sequence are mixed with a fixed amount of PM and the reactions are subject to PCR.
  • the concentration of the target sequence is determined by comparing the yield of PCR products obtained with a calibration curve constructed at the same time using material containing known amounts of the target sequence.
  • the present inventors have now determined the primary structure of the LSU rDNA of N. caninum. Comparison with a consensus sequence derived for the LSU rDNA of T. gondii demonstrated that the D2 domain
  • Neospora can serve as a target for the development of a species-specific PCR for the detection of rDNA from Neospora.
  • the present inventors have now identified a new genetic marker that can be vised to distinguish between Neospora spp and Toxoplasma spp.
  • useful PCR MIMICs have been developed for use in cPCR assays for Neospora. Disclosure of Invention
  • the present invention consists in an isolated nucleic acid molecule encoding the large subunit (LSU) ribosomal DNA (rDNA) of Neospora spp, the nucleic acid molecule having a sequence as set out in Figure 2, or a functionally equivalent sequence thereof, or a sequence that hybridises thereto.
  • LSU large subunit
  • rDNA ribosomal DNA
  • the Neospora spp is Neospora caninum.
  • sequences which hybridise to the sequence shown in Figure 2 hybridise under stringent conditions.
  • stringent conditions are those that (a) employ low ionic strength and high temperature for washing, for example, 0.015 M NAC1/0.0015 M sodium citrate/0/1% NaDodS0 4 at 65°C; (b) employ during hybridisation a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C; or (c) employ 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardfs solution
  • the present invention consists in nucleic acid primers which are complementary to and specific for the LSU ribosomal DNA of Neospora spp.
  • the primers are used in a polymerase chain reaction (PCR) assay, the primers bind to LSU ribosomal DNA of Neospora spp present in a sample so as to allow the amplification of a nucleic acid molecule according to the first aspect of the present invention.
  • PCR polymerase chain reaction
  • the primers include the sequences selected from: NF6 5'-GTCCCTCGTGGACCC; and
  • the primers are NF6 and GAl.
  • the present invention consists in nucleic acid primers useful for the detection of Neospora spp by PCR in assays of clinical specimens, the primers including the sequences selected from: TimllF 5'-GGTACGTCTGTTTCAGTG; Timl ⁇ 5'-CTCTCTCACCAGGTTTAG; Timl2 5'-GACCTAAAGGATCGATAG;
  • the primers are TimllF, Timl5, Timl2, GA5, or GA6.
  • TimllF and GAl are particularly suitable to amplify the LSU rDNA of Neospora spp using PCR.
  • the present invention consists in a unique Neospora species-specific PCR product of 270 bp obtained using the primer GAl with primer NF6 under standard PCR conditions.
  • the present invention consists in a method of obtaining an isolated nucleic acid molecule encoding the LSU ribosomal
  • DNA of Neospora spp the method including amplifying the nucleic acid molecule by PCR using primers according to the second or third aspects aspect of the present invention.
  • the primers according to the second or third aspects of the present invention can be used to identify or diagnose the presence of Neospora spp in a sample using PCR.
  • an assay based on PCR which is sensitive and specific for Neospora spp can be developed.
  • the assay has the potential to provide the differential diagnosis between Neospora spp and Toxoplasma spp or other related or non-related microorganisms.
  • the present methods preferably require a nucleic acid molecule containing LSU sequence flanked by parasite-specific primers to be synthesised and cloned into a plasmid vector such as pGEM-T.
  • the recombinant molecule may then be produced in large quantities using standard genetic engineering techniques for plasmid production, purified and seeded into a PCR reaction at known concentration in order to act as an internal standard for the PCR. Knowing the full sequence information of Neospora spp LSU has allowed the generation of suitable positive control internal sequences for use in PCR tests.
  • the present invention consists in an internal reference standard, competitor sequence or PCR MIMIC for use in a Competitive PCR (cPCR) for Neospora spp, the internal reference standard, competitor sequence or PCR MIMIC having a nucleotide sequence including the following priming sites:
  • IP1 S'-CATGTGGATATTTTGCAGTCCCTCGTGGACCC and
  • the internal reference standard, competitor sequence or PCR MIMIC has the following sequence:
  • the internal reference standards, competitor sequences or PCR MIMICs according to the fifth aspect of the present invention can be included in the method according to the fourth aspect of the present invention.
  • the present inventors have also devised internal reference standards.
  • competitor sequences or PCR MIMICs for T. gondii These internal reference standards, competitor sequences or PCR MIMICs can be included in PCR assays for Neospora spp and will allow ability to differentiate between
  • Neospora spp and T. gondii The sequences are as follows: a) priming sites IP4 and IP4
  • IP3 (5'-TCCATTGGAGAGATTTGCGTTCCTTGTGGACCG); and IP4 (5'-AAACTCCTGGAAATCAGTAAACCTCTCTCAGAGATCG).
  • IP4 5'-AAACTCCTGGAAATCAGTAAACCTCTCTCAGAGATCG.
  • sequences including the primer pairs could be developed from the sequences provided.
  • sequences including the primer pairs could be developed from the sequences provided.
  • sequences including the primer pairs could be developed from the sequences provided.
  • sequences including the primer pairs could be developed from the sequences provided.
  • sequences including the primer pairs could be developed from the sequences provided.
  • sequences including the primer pairs could be developed from the sequences provided.
  • sequences including the primer pairs could be developed from the sequences provided.
  • IP1/IP2 having positioned therebetween an amplifiable string of nucleotides would also be suitable for this purpose.
  • Additional internal reference standards, competitor sequences or PCR MIMICs may also include nucleotides positioned at the 5' and/or 3' ends of the primer pairs IP1/IP2. It will also be appreciated that internal standards for other microorganisms including Hammondia liammondi can be devised and used in PCR assays for Neospora spp.
  • Figure 1 shows the location of primer sequences used for the PCR isolation of the LSU rDNA of N. caninum or T. gondii. Primer sequences are given in the Materials and Methods. Arrows indicate the direction of the primer extension reaction.
  • Figure 2 shows the sequence of an isolated nucleic acid molecule encoding the LSU ribosomal DNA of Neospora spp.
  • FIG. 3 shows the comparison of the LSU rDNA of N. caninum and T. gondii.
  • the LSU rDNA of N. caninum (N.c.) was aligned with that of T. gondii (GenBank accession number L25365). Only bases which are different between the sequences are indicated. In regions where the four T. gondii sequences disagree (GenBank accession numbers L25365, X75429, X75430 and X75453), a consensus sequence was generated (T.g.).
  • ![Timl5/GAl] represents the fragment that was independently amplified by PCR and sequenced from Neospora (NCI, NC-Liverpool and SweBl strains) and
  • Toxoplasma (RH, ME49 strains) and compared to X75429, X75430 and X75453.
  • 2 ⁇ deletion ⁇ represents the region of rDNA that is deleted in X75429.
  • 3 ⁇ replacement ⁇ represents the region of rDNA that is replaced in sequence X75430. Numbers given at the beginning and end of the sequence alignment indicate the base position in the alignment. Areas of inferred rRNA secondary structure which are discussed in the text are indicated between
  • Tachyzoites of N. caninum (NC-Liverpool strain) or T. gondii (RH strain) were propagated by in-vitro culture on a Vero monolayer as described [4).
  • Tachyzoites were purified by filtration through a 3 micron nucleopore filter [7].
  • DNA was prepared from tachyzoites by standard procedures involving lysis in a Tris buffer containing SDS, EDTA and proteinase K, followed by phenol/chloroform extraction and ethanol precipitation. Extraction of DNA
  • Genomic DNA was purified from 10 8 tachyzoites by lysis in 1% SDS, 100 mM EDTA, 10 mM Tris pH 9 containing 100 ⁇ g/ml proteinase K at 56 °C for 2 hours. DNA was further purified by phenol/chloroform extraction; centrifiigation through a cesium chloride gradient (density 1.68 gm per ml); followed by dialysis against 10 mM Tris pH 7.5, 0.1 mM EDTA.
  • LSU rDNA of N. caninum was isolated as a series of overlapping fragments by PCR ( Figure 1). PCR fragments were obtained by primer combinations TimllF (5'-GGTACGTCTGTTTCAGTG) and GAl (5'-AACCTCTCTCAGAGATCG); Timl ⁇ (5'-CTCTCTCACCAGGTTTAG) and GAl;
  • Tim ⁇ and Timl2 (5'-GACCTAAAGGATCGATAG); and GA5 (5 1 - CT ⁇ TCGATCCTTTAGGTC) and GA6 ⁇ '-GCACGTGCACTCCGCATTTG.
  • PCR products were purified by a QIAquick purification column (Qiagen, USA); ligated into the plasmid vector pGEM-T (Promega, USA) and transformed into Escherichia coli (DH ⁇ alpha).
  • Transformants containing insert DNA were identified by growth on L-agar plates containing ampicillin, IPTG and Xgal; grown in L-broth containing ampicillin and plasmid DNA prepared from them using the Qiagen miniprep kit. Inserts were sequenced in both directions by cycle sequencing (Sequitherm, Epicentre, USA) using IRD-41 fluorescein labelled M13 forward or reverse primers (LiCOR, USA).
  • the products of the cycle sequencing were run and visualised on a LiCOR 4000L automated DNA sequencing machine. At least 6 clones were sequenced in both directions for each PCR product cloned (i.e. 12 sequencing reactions in total) and a consensus sequence was obtained for each fragment using AssemblyAlignTM . A consensus sequence for the LSU rDNA was then obtained by compiling the sequence from the overlapping fragments. Using this approach 3,499 nucleotides of the LSU rDNA of N. caninum were obtained. During early studies on comparing LSU rDNA from N. caninum and T gondii, comparisons were made using the L2563 ⁇ entry for the RH strain [2] since this represents the only sequence of T.
  • Tim3 anneals to the 3 1 end of the SSU while Tim6 anneals to the 5S rDNA. Both Tim3 and Tim6 have Pstl sites at their 5' ends and these Pstl sites were used to clone the PCR product into the plasmid vector pUCl9.
  • the ligation products were electroporated into E. coli (strain DH ⁇ alpha) and transformants were selected on L- agar plates containing ampicillin, IPTG and Xgal. Individual white colonies were cultured in liquid media, plasmid DNA isolated and the insert DNAs were examined by restriction analysis using Pstl. One positive clone obtained from this process was selected and called pRHT3T6. This single clone was sequenced by cycle sequencing and an ABI automated sequencer using a combination of the primers described above.
  • the primers Timl ⁇ and GAl were used to amplify a DNA fragment covering the D2 and D3 expansion segments of the LSU rDNA from RH and ME49 strains of T gondii; NCl and NC-SweBl strains of N. caninum and
  • Neospora and Toxoplasma were grown in-vitro and DNA prepared as described above.
  • the PCR products were cloned into pGEM-T, sequenced and aligned to available data for N. caninum (NC-Liverpool) and T gondii (X7 ⁇ 429, X7 ⁇ 430 and X7 ⁇ 453) by Clustal W.
  • a comparison of the sequences derived from the D2 expansion segment (Cl/Cl' region) of Neospora and Toxoplasma revealed nucleotide sequences which were incorporated into a species-specific primer designed for N caninum (NF6; 5'-GTCCCTCGTGGACCC).
  • a similar primer was designed against the T gondii sequence (TF6; ⁇ '-GTTCCTTGTGGACCG) but subsequently proved not to be species-specific.
  • Neospora, Toxoplasma, or Hammondia DNA was performed using either NF6 or TF6 with GAl.
  • Toxoplasma DNA from 23 independent strains (21 of them are described in [6]; RH and RH88 DNAs are described in [ ⁇ ]) were subject to PCR using primers GA7 ( ⁇ '-ATTCGCTTTACCTGA) and GA8 (5'-
  • IPl represents a composite of primers NS2 ( ⁇ '-CATGTGGATATTTTGCA) and NF6 ( ⁇ '-GTCCCTCGTGG ⁇ CCC) whereas primer IP2 represents a composite of primers NRl (5'-AAACTCCTGGAAGTTAAAG) and GAl ( ⁇ '-AACCTCTCTCAGAGATCG). Under these conditions, priming sites for NS2 and NRl are placed on either side of a 270 bp fragment derived from the
  • D2 domain of the large subunit (LSU) ribosomal (r) DNA (which is amplified by primers NF6 and GAl).
  • the 306 bp PCR product generated was purified using a Qiagen column and the sequence determined by cycle sequencing using primer NS2 or NRl.
  • the PCR product was cloned into the plasmid vector pGEM-T and transformed into Escherichia coli by standard calcium chloride transfection techniques. Plasmid DNA was extracted from recombinant bacterial clones by the procedure of alkaline lysis, purified by a Qiagen column, and the DNA concentration determined by optical density.
  • IP3/IP4 A competitor molecule (hereafter called IP3/IP4) was also made using the process described here from primers IP3
  • IP3 represents a composite of primers TS4 (5'-TCCATTGGAGAGATTTGC) and TF6 (5'- GTTCCTTGTGGACCG) whereas primer IP4 represents a composite of primers TRl ( ⁇ '-AAACTCCTGGAAATCAGTA) and GAl ( ⁇ '-AACCTCTCTCAGAGATCG).
  • IP3/IP4 is, therefore, compatible with the T. gondii specific primers TS4 and TRl and PCR amplification of IP3/IP4 with TS4 and TRl generates a PCR product of 307 bp.
  • reaction conditions for the cPCR comprised ⁇ ⁇ l of test sample; lxPCR Buffer; 1.7 ⁇ mM MgCl 2 ; ⁇ 0 ⁇ M of NS2 and NRl (or TS4 and TRl) and
  • PCR was performed on a Hybaid omnigene with the following thermal cycle program: 9 ⁇ °C for 5 minutes; 5 cycles of 94°C for 30 seconds, 60°C for l ⁇ O seconds, 72°C for 30 seconds; l ⁇ cycles of 88°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds; 10 cycles of 88°C for 30 seconds, ⁇ 4°C for 30 seconds, 72°C for 30 seconds; 2 ⁇ cycles of 86°C for 30 seconds, 54°C for 30 seconds, 72°C for 30 seconds; and finally 72°C for 10 minutes.
  • NS2 and NRl generates a PCR product of 146 bp from N. caninum genomic DNA
  • TS4 and TRl generates a PCR product of 18 ⁇ bp from T gondii DNA.
  • a hot start PCR was used to prevent PCR amplification of non-specific products.
  • the products of the PCR were run on a 2% agarose gel which included either a 100 bp molecular weight ladder or a Hpa ⁇ l digest of pUCl3 as markers.
  • tachyzoites were concentrated by centrifugation and counted using a hemocytometer.
  • Female, in-bred Balb/C mice sourced from ARC, Perth, Western Australia or Gore Hill Research Laboratories, NSW) approximately 20 g in body weight, were housed in groups of 6-12 in plastic box cages and provided feed and water ad libitum.
  • doses of tachyzoites were inoculated subcutaneously into mice.
  • the brains of N. caiunum infected mice were removed at either 9, l ⁇ or 18 days post infection (dpi); DNA was prepared from them using a QIAamp tissue Kit (Qiagen) as recommended by Jenkins et al. [8] and subject to cPCR using primers NS2 and NRl in the presence of the IP1/IP2 PM. Brains from uninfected mice were used as negative controls.
  • DNA was then used in a cPCR.
  • gondii sequences in this region were the result of transitions (either T>C or C>T), compared to one transversion (C>G).
  • C>G the sequence of this region for the bovine strain of N. caninum (NC-SweBl) is identical to the other two strains of N. caninum isolated from dogs (NCI and NC-Liverpool).
  • PCR primers NF6 and TF6 were designed based on the observed nucleotide differences between Neospora and Toxoplasma in the Timl5/GAl sequence in an attempt to generate species-specific PCR primers.
  • the primer combination GAl and NF6 under PCR conditions that utilise a primer annealing temperature of 5 ⁇ °C produce a unique species-specific PCR product of 2 ⁇ 0 bp from N. caninum DNA. No product is obtained using these two primers with Toxoplasma, or Hammondia DNA unless the primer annealing temperature is lowered to ⁇ 2°C.
  • the primer combination TF6 and GAl generated no PCR products from Neospora DNA using a primer annealing temperature of 55°C.
  • Neospora spp In order to ensure that the PCR reaction has not failed during a diagnostic procedure, a positive control is necessary for inclusion as an internal standard. Knowing the full sequence information of Neospora spp
  • LSU allowed the present inventors to the generate suitable positive control internal sequences for use in PCR tests.
  • IPl ⁇ '-CATGTGGATATTTTGCAGTCCCTCGTGGACCC
  • IP2 ( ⁇ '-AAACTCCTGGAAGTTAAAGAACCTCTCTCAGAGATCG); IP3 ( ⁇ '-TCCATTGGAGAGATTTGCGTTCCTTGTGGACCG); and IP4 ( ⁇ '-AAACTCCTGGAAATCAGTAAACCTCTCTCAGAGATCG).
  • IP1/IP2 and IP3/IP4 are shown below. DNA Sequences of the PCR MIMICs developed are described below: a) DNA sequence of IP1/IP2 PCR product ⁇ 'CATGTGGATATTTTGCAGTCCCTCGTGGACCCTTATATCTTTGTTCTTTC CTTTTCCTTGTGGCTGAGGAGTGTTCTTGTTTCCGAGCTCCACTTTCGAGT ACTCGGTTTCTGTGATGCTGGCTTAATCGGTTCCAACCGACCCGTCTTGA AACACGGACCAAGGAGTCTAACATATGTGCGAGTATGCGGGTTTTACTCC TGTATGCGCAATGAAAGTGAGAGTAGGGAGATTTTGGCTTTGCCATTCTT CGCACCTACGACCGACCACGATCTCTGAGAGAGGTTCTTTAACTTCCAGG AGTTT b) DNA sequence of IP3/IP4 PCR product
  • IP1/IP2 As a PM for a cPCR assay, 30 pg of IP1/IP2 were added to individual reactions containing serial dilutions of genomic DNA from N. caninum and subject to PCR. The products of the cPCR were subject to agarose gel electrophoresis. Two bands were detected on the gel, the largest derives from the PM whereas the smaller one derives from the genomic DNA template. The yield of the two bands was dependent on the amount of genomic DNA included in the PCR reaction. In another experiment, each lane of the gel was scanned using a densitometer and the natural log of the relative density of the two PCR products in each track were plotted against the mass of the target sequence present in the PCR reaction.
  • a calibration curve demonstrated an equivalence point reached with approximately 50 pg of IP1/IP2 and 800 ng of genomic DNA, implying both populations of DNA contain similar numbers of priming sites (e.g. that 0.05/800 or approximately 0.2 % of N. caninum genomic DNA represents ribosomal DNA).
  • a cPCR containing target and PM should fulfil the principle condition that the two sequences are PCR amplified with equal efficiencies by identical primers so that a plot of log (T 0 /C 0 ) against log C 0 should be a straight line with a gradient of -1 [9] . Since this issue appears to be generally ignored by most workers, data generated for IP1/IP2 was subject to analysis.
  • a calibration curve of log (T 0 /C 0 ) against log C 0 showed linearity over the range of 5 to 3000 ng of genomic DNA. The shape of the curve was therefore consistent and compatible with its use as a calibration curve for the prediction of DNA concentrations in biological specimens. The range of linearity could be modified however, by varying the amount of PM included in the reaction.
  • mice Since one aim of these experiments was to design a cPCR which could be used to detect Neospora DNA in the brains and fetal tissues, for example, of animals such as mice, the effect of mouse brain DNA on the PCR was investigated. An excess of normal mouse brain DNA (0, 100 ng, 1 or ⁇ ⁇ g) was mixed with 2 ⁇ 6 pg of N. caninum DNA and 30 pg of PM and subject to PCR. Using these masses of PM and Neospora genomic DNA , it was predicted from experiments that only a 146 bp product from the Neospora genomic DNA should be generated. The control experiment where no mouse brain DNA was incorporated shows this to the case.
  • mice were infected with culture-derived tachyzoites.
  • NC- Liverpool induced severe clinical signs of neosporosis in the infected mice including discoordinated movement, hindlimb paralysis and coat ruffling with severe weight loss at days 17-28 after inoculation.
  • DNA from the brains of mice sacrificed at 9 dpi did not support the generation of a 146 bp PCR product by cPCR using 30 ng PM; a 146 bp PCR product was produced however in the presence of the PM at l ⁇ (in mice infected with 10 6 tachyzoites) and 18 (in mice infected with 10 5 tachyzoites) dpi.
  • the primary structure of the LSU rDNA of N. caninum has been determined by PCR and DNA sequencing and it is highly similar to the LSU rDNA of T gondii. In T. gondii Gagnon et al. (1996) [2] recognised 81 helices and 12 expansion segments (called Dl to D12) in the 28S rRNA. No structure was suggested for the expansion segments. Similarly the Antwerp LSU ribosomal database contains a secondary structure prediction for T gondii LSU rRNA that contains at least 102 helices and presents a proposal for the secondary structure for the total LSU rRNA [1].
  • L2536 ⁇ contained 26 bases in the region between the stems of D ⁇ and D ⁇ ' whereas the N. caninum and T. gondii sequences X7 ⁇ 429, X7 ⁇ 430 and X7 ⁇ 4 ⁇ 3 contain 27 bases.
  • the sequences of D14 were inconsistent and the base pairing suggested by them were not consistent with the secondary structure model described in [1].
  • the sequence of L25365 was missing TT in Dl ⁇ , whereas the sequence determined for N.
  • the LSU rRNA is the largest of the rRNAs, yet it has not been used extensively for studies on the phylogeny of parasites because few sequences are available and because their sequences probably have rates of evolution similar to those of the SSU rRNA.
  • Specific regions (primarily the expansion or variable domains) of the LSU sequences have proven useful for distinguishing among a number of proposed alternatives of vertebrate phylogeny.
  • the observations that the expansion segment D2 contains nucleotides that differ between Neospora and Toxoplasma shows that this region may be useful for investigating the relationships among these cyst- forming coccidia.
  • N. caninum and T gondii provides further evidence for a close taxonomic relationship between these two taxa.
  • Toxoplasma possesses a facultative heteroxenous life cycle whereas Hammondia is obligatory heteroxenous, and the cyst stages are found predominantly in skeletal muscle of the intermediate host in Hammondia and not in neural tissue.
  • Cl/Cl 1 refers to D2 domain [2].
  • Competitor molecules were constructed by PCR using primers that were composites of two primers. Species-specific priming sites normally located only in the ITSl were introduced onto either side of a small fragment of the LSU rDNA, resulting in the generation of a synthetic competitor molecule that could be amplified using the primers directed against the ITSl. Two PMs were made: IP1/IP2 containing Neospora LSU rDNA flanked by primers that amplify specifically Neospora ITSl (NS2 and NRl); and IP3/IP4 containing Toxoplasma LSU rDNA flanked by primers specific for the ITSl of
  • T gondii (TS4 and TRl).
  • a calibration curve of log (T 0 /C 0 ) against log C 0 showed linearity over the range of ⁇ to 3000 ng of genomic DNA using the cPCR described thus confirming the utility of the PM and the cPCR. Outside of this, an excess of either target or competitor saturated the PCR. Therefore ⁇ to 3000 ng of genomic DNA represents the range over which quantitation of target sequence can be reliably performed using the conditions described, if the PCR products are analysed by agarose gel electrophoresis and ethidium bromide staining. This represents approximately ⁇ x 10 4 to 3xl0 7 tachyzoites.
  • PCR products can be detected and quantified (e.g. incorporation of radioactive label into the PCR and autoradiography) that may potentially alter the range over which the competitive PCR is functional.

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  • Biophysics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne une molécule d'acide nucléique isolée codant pour l'ADN ribosomique à sous-unités de grande taille (ADNr) de l'espèce Neospora, ladite molécule d'acide nucléique possédant une séquence représentée dans la Figure 2, ou une séquence équivalente de celle-ci, ou une séquence qui s'hybride à celle-ci; et des procédés de détection de l'espèce Neospora au moyen de l'amplification en chaîne par polymérase (PCR).
PCT/AU1998/000367 1997-05-20 1998-05-19 Adn ribosomique a sous-unites de grande taille, de l'espece $i(neospora) Ceased WO1998053074A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU74188/98A AU7418898A (en) 1997-05-20 1998-05-19 Large subunit ribosomal dna of (neospora) species

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AUPO6903 1997-05-20
AUPO6903A AUPO690397A0 (en) 1997-05-20 1997-05-20 Large subunit ribosomal dna of neospora species

Publications (1)

Publication Number Publication Date
WO1998053074A1 true WO1998053074A1 (fr) 1998-11-26

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU1998/000367 Ceased WO1998053074A1 (fr) 1997-05-20 1998-05-19 Adn ribosomique a sous-unites de grande taille, de l'espece $i(neospora)

Country Status (2)

Country Link
AU (1) AUPO690397A0 (fr)
WO (1) WO1998053074A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6600027B1 (en) 1998-03-26 2003-07-29 Pfizer, Inc. Polynucleotide molecules encoding neospora proteins
CN111197100A (zh) * 2020-01-14 2020-05-26 中国农业大学 犬新孢子虫特异pcr检测试剂盒及制备方法和应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997027300A1 (fr) * 1996-01-26 1997-07-31 Innogenetics N.V. Antigene tg20 de toxoplasma gondii
WO1998008970A1 (fr) * 1996-08-26 1998-03-05 Insearch Limited Detection de its1 dans toxoplasma gondii et neospora caninum par pcr

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997027300A1 (fr) * 1996-01-26 1997-07-31 Innogenetics N.V. Antigene tg20 de toxoplasma gondii
WO1998008970A1 (fr) * 1996-08-26 1998-03-05 Insearch Limited Detection de its1 dans toxoplasma gondii et neospora caninum par pcr

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
EMBL, Accession No. X75430, X75429, Published November 1993. *
GENE, Vol. 173, (1996), GAGNON et al., "Secondary Structures and Features...", pp. 129-135. *
INT. J. PARISTOL., Vol. 26(4), (1996), PAYNE et al., "Detection of Neospora Caninum DNA by the Polymerase Chain Reaction", pp. 347-351. *
MOLECULAR AND CELLULAR PROBES, Vol. 10, (1996), KAUFMANN et al., "Discrimination of Neospora Caninum ...", pp. 289-297. *
MOLECULAR AND CELLULAR PROBES, Vol. 12, (1998), ELLIS et al., "Comparison of the Large Subunit Ribosomal DNA of Neospora ...", pp. 1-13. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6600027B1 (en) 1998-03-26 2003-07-29 Pfizer, Inc. Polynucleotide molecules encoding neospora proteins
CN111197100A (zh) * 2020-01-14 2020-05-26 中国农业大学 犬新孢子虫特异pcr检测试剂盒及制备方法和应用

Also Published As

Publication number Publication date
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