EP1012337A1 - Diagnosis of ocular pathogens - Google Patents

Diagnosis of ocular pathogens

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Publication number
EP1012337A1
EP1012337A1 EP98942853A EP98942853A EP1012337A1 EP 1012337 A1 EP1012337 A1 EP 1012337A1 EP 98942853 A EP98942853 A EP 98942853A EP 98942853 A EP98942853 A EP 98942853A EP 1012337 A1 EP1012337 A1 EP 1012337A1
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Prior art keywords
oligonucleotide primers
primers
bacterial
seq
pcr
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EP98942853A
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German (de)
French (fr)
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Narciss Okhravi
Susan The Institute of Ophthalmology LIGHTMAN
Peter The Institute of Ophthalmology ADAMSON
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Institute of Ophthalmology UCL
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Institute of Ophthalmology UCL
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    • 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
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • 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
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to the diagnosis of ocular pathogens.
  • Ocular infection can result in a profound loss of vision.
  • Infection of the cornea can be broadly divided into that caused by bacteria, fungi and Acanthamoeba species, whereas intraocular infection (endophthalmitis) can be either bacterial or fungal.
  • Table 1 illustrates the most common ocular pathogens.
  • Fungal endophthalmitis is one example of ocular infection and accounts for 4-11% of all cases of culture proven endophthalmitis. It is usually acquired from an endogenous source but may also be secondary to intraocular surgery, corneal ulceration or trauma. The time to diagnosis from the onset of symptoms has been reported as varying from 3 days to 4 months. Fungal endophthalmitis may have a classical appearance e.g Candida albicans, but it may also present in a similar manner to chronic bacterial endophthalmitis, or it may coexist with bacterial endophthalmitis. Although C. albicans is the causative agent in the majority of cases of culture-proven fungal endophthalmitis, C. tropicalis, C. krusei, C.
  • glabrata, and C. parapsilosis are increasingly recognised as capable of causing endophthalmitis.
  • these isolates eg. C. glabrata, and C. krusei
  • non-albicans spp as ocular pathogens is of particular concern as very few drugs are available which, when given systemically, achieve adequate vitreous concentrations even in eyes with inflamed blood-ocular barriers.
  • the drugs fluconazole and flucytosine achieve good penetration into the central nervous system and ocular tissues.
  • Amphotericin B remains the only drug to which resistance has not been encountered in ocular isolates. This drug, however, requires intraocular injection as intravenous administration achieves adequate concentrations only in the retina and choroid but not in the vitreous.
  • awareness of possible drug resistance to fluconazole is essential. Should resistance occur, the patient will not be receiving the appropriate systemic antifungal therapy to continue treatment for the diseased eye; to treat or protect the fellow eye; or to combat systemic disease.
  • PCR polymerase chain reaction
  • the present invention relates to a method for the rapid diagnosis of the presence or absence of ocular infection, and the identification of pathogens to the species level.
  • the present invention discloses a method for determining the presence of ocular pathogens in a sample, comprising the steps of:
  • step (ii) amplifying the nucleic acids using a set of bacterial, fungal or Acanthamoeba -specific oligonucleotide primers; (iii) amplifying the product of step (ii) using nested or semi-nested bacterial, fungal or Acanthamoeba-specific oligonucleotide primers; and (iv) detecting any second amplified product.
  • the method is very sensitive and a diagnosis can be made for fungi even if only one gene copy is present and for bacteria if only one organism is present. Furthermore, the method significantly reduces the time taken to make a diagnosis.
  • the invention is based on the finding that nested or semi-nested oligonucleotide primers can be used to improve sensitivity in the amplification of pathogen DNA from an ocular sample. Two amplification steps are required: the first requires oligonucleotide primers having broad specificity for ocular pathogens; the second uses nested or semi- nested primers to re-amplify the first step products and to provide genus-specific and in some instances species-specific information. The further use of restriction enzymes may provide species-specific information in the majority of cases.
  • the first amplification step uses a pair of oligonucleotide primers designed to amplify highly conserved gene sequences.
  • the primer pairs for bacterial pathogens, fungal pathogens and Acanthamoeba species may initially be used together to indicate whether or not an ocular pathogen is present in a sample.
  • the products from this first amplification step are then re-amplified using nested or semi-nested oligonucleotide primers. It is preferable that these primers also amplify highly conserved gene sequences of bacteria, fungi and Acanthamoeba and are genus- specific rather than species-specific. It is therefore possible to identify broadly the organisms most likely causing infection, i.e. either bacterial, fungal or Acanthamoeba species, immediately following the second amplification step.
  • the products from either the first or second step can be treated with a cocktail of restriction enzymes.
  • Each species will have a particular restriction enzyme map. By using various combinations of restriction enzymes, it may be possible to identify the different species present on the basis of their individual restriction enzyme maps.
  • Intraocular sampling is routinely undertaken on all patients with suspected endophthalmitis.
  • the extraocular environment was sterilised with 5% Povidone Iodine solution prior to surgery.
  • Approximately 100-200 ⁇ l of aqueous fluid was withdrawn using a 27G (0.33mm) needle via a limbal paracentesis.
  • Vitreous samples 200-400 ⁇ l were taken as a biopsy through the pars plana.
  • Ocular samples were examined by Gram and Giemsa staining, and cultured on blood agar, Sabauraud's agar, in Brain Heart Infusion (BHI) and Robertson's Cooked Meat Broth (CMB). Cultures were incubated under both aerobic and anaerobic conditions. All cultures were maintained for 14 days. Isolates were maintained at -70°. Procedure for keratitis cases
  • Corneal scrapes were performed at the slit lamp under topical anaesthesia. Sterile 27G needles were used to scrape off sections of corneal epithelium and anterior stroma in the region of the leading edge of the corneal ulcer. Samples were placed on a slide for Gram and Giemsa staining and also plated immediately on Blood and Sabauraud's non- nutrient agar. Plates were incubated under aerobic conditions at 25°C, 30°C or 37°C as appropriate.
  • Vitreous was collected by sterile technique at the time of vitrectomy during planned surgical procedures in patients with no evidence of intraocular infection or inflammation or medical history of uveitis and/or diabetes mellitus. Samples of normal vitreous were aliquoted in a sterile manner and stored at -20 °C. DNA extraction 1) Fungi Method 1: Full extraction procedure
  • DNA was extracted by a method which was modified from that described by Buchman et al, Surgery (1990) 108:338-347.
  • DNA extraction tubes were prepared as follows: each tube contained 5 ⁇ 1 of neat vitreous, 100 ⁇ l of 0.05 mol/1 Tris pH 7.5, 0.01 mol/1 EDTA, 0.028 mol/1 ⁇ -mercaptoethanol containing 0.3mg/ml zymolase and 5 ⁇ l of the required dilution of C. albicans in water. Samples were incubated for 30 minutes at 37°C, followed by addition of 0.1% SDS and 15 ⁇ g/ml proteinase K, and incubation continued for a further 5 minutes.
  • the mixture was subsequently heated to 95 °C for 5 minutes and cooled on ice for 15 minutes.
  • Samples were extracted with phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.0), chloroform and precipitated with 2.5 volumes of ethanol in the presence of 300mM sodium acetate.
  • the DNA was pelleted at 14,000g for 20 minutes, washed with ice cold 80% ethanol, air dried at 65 °C for 10 minutes and resuspended in 25 ⁇ l of sterile TE buffer (lOmM Tris, ImM EDTA, pH8.0).
  • Method 2 Boil and PCR
  • the DNA extraction tubes were prepared as follows: each tube contained 5 ⁇ l of neat vitreous and 5 ⁇ l of the required dilution of C. albicans in water. Samples were incubated at 95 °C for 20 minutes and subsequently the entire volume was used in PCR reactions. 2) Bacteria Method 1: Full extraction procedure
  • primers used to amplify a highly conserved gene sequence in a pathogen appear in Table 2.
  • a suitably conserved gene is the 16S rDNA gene.
  • primers to the cytochrome P 450 L ⁇ demethylase gene can be used.
  • the 18S ribosomal gene may be used for fungal pathogens. Further embodiments of the invention are as described in the sub-claims.
  • Other suitably conserved genes that may be used as targets for amplifying yeast, bacterial ox Acanthamoeba DNA, will be apparent to those skilled in the art.
  • Replitherm Taq (Cambio) DNA polymerase was used for bacterial PCR, whereas, Amplitaq (Perkin-Elmer) DNA polymerase was used for yeast and Acanthamoeba PCR.
  • Optimisation for annealing temperature, and magnesium concentration can be carried out to find the primer sets which produce the highest product yield and greatest sensitivity whilst retaining high specificity.
  • Example 1 The following examples illustrate the invention with respect to the identification of fungal pathogens (Example 1) and bacterial pathogens (Example 2).
  • Example 1 The following examples illustrate the invention with respect to the identification of fungal pathogens (Example 1) and bacterial pathogens (Example 2).
  • Both first round and second round reactions contained 100 ⁇ M each dNTP (Pharmacia), 200 ⁇ M of each primer, 2.5mM magnesium chloride, 1 unit DNA polymerase lOOmM Tris-HCl ( pH 8.3) 500mM KC1; 15mM MgCl 2 ; 0.001%w/v gelatin, in a 50 ⁇ l reaction. Cycling conditions were identical for both rounds except for the annealing temperature which was found to be optimal at 51.5 ° C for the first round (primers SEQ ID NO: 7 & 8), 56°C for nested primers I (SEQ ID NO: 9 & 10) and 58 °C for semi-nested primers II (SEQ ID NO: 9 & 8).
  • PCR cycling was performed at 95 °C for 10 minutes for one cycle, followed by 30 cycles of 94 ° C for 1 minute, annealing temperature as above for 1 minute, and extension at 72°C for 1 minute. The final cycle was followed by 10 minutes at 72 °C.
  • PCR products were resolved on a 1-2 % agarose/TBE gel according to the size of fragment expected and visualised using ethidium bromide under UV illumination.
  • Restriction enzyme digests were performed at 37°C for 2 hours in buffer containing 6mM Tris -HC1, 6mM Mg Cl 2 , 150 mM NaCl at pH7.9 and 10 units of each enzyme. The resulting fragments were subsequently resolved on 4-8% polyacrylamide gels and visualised under UV illumination following staining with ethidium bromide.
  • the sensitivity of the first round PCR was routinely found to be 30-300 pg from dilutions of DNA from C. albicans starting from a concentration of lOng/ ⁇ l. Assuming a total DNA content of 37fg per organism, and two copies of the gene per diploid genome this is equivalent to 800-8000 organisms.
  • the second round of PCR markedly improves this sensitivity to 10 fg which is equivalent to less than one gene copy.
  • lOfg is less than the concentration of DNA in one organism, we proceeded to PCR amplify 1 Ofg of C. albicans genomic DNA twenty times expecting a positive result one time out of three.
  • a fresh DNA dilution series was prepared using 1.5 ⁇ g of C.
  • albicans genomic DNA (a concentration at which spectrophotometric DNA concentration measurements are reliable).
  • PCR-RFLP was performed on eight Gram positive and six Gram negative organisms known to be causative agents of endophthalmitis. Two pairs of oligonucleotide primers based on the 16s rDNA gene were used to PCR amplify a 1.2 kb and a 1.0 kb fragment of bacterial genomic DNA. Restriction fragment length polymorphisms within the PCR product were identified following sequence analysis of the amplified products and subsequently used to speciate the organisms following restriction analysis.
  • 16s rDNA genes are ubiquitous and are present as multiple copies within bacterial genomes. Optimisation for annealing temperature, and magnesium ion concentration were undertaken in an attempt to find the experimental conditions which produced the highest product yield and greatest sensitivity whilst retaining high specificity. Optimal PCR conditions
  • PCR reactions contained 20mM Tris-HCl (pH8.3), 100 mM KC1, 0.01% Tween 20, 0.1% NP-40, lOO ⁇ M each deoxyribonucleotide ('dNTP', Pharmacia Biotech, St Albans, UK), 0.3 ⁇ M of each primer, 1.5mM magnesium chloride, 3 units Replitherm Taq DNA polymerase ('Taq', Cambio Ltd. Cambridge, UK), in a 25 ⁇ l reaction. Both first round and nested PCR cycles commenced with 5 minutes at 95 °C.
  • the Taq DNA polymerase used in this study is one of a number of commercially available Taq polymerases with known low levels of DNA contamination. Although this level of contamination is insufficient to give a detectable amplification product after just one round of PCR, it is easily detected following nested amplification. Therefore, in this study, prior to first round PCR amplification, the Replitherm Taq was treated with Alul restriction endonuclease (ratio of 3 : 1 units of Taq: Alul, Promega UK Ltd., Southampton, UK), which recognises a specific nucleotide sequence AGCT.
  • this restriction enzyme was firstly its known high cutting frequency within the 16s rDNA genomic template, which was evident following sequence analysis of the amplified fragments from the fourteen bacterial species, and secondly the ability to heat inactivate the enzyme prior to addition of template DNA.
  • PCR buffer, magnesium and Taq components Prior to PCR amplification the water, PCR buffer, magnesium and Taq components were mixed and incubated at 37°C with Alul for 30 minutes.
  • the restriction enzyme was subsequently inactivated by incubation at 95 °C for two minutes, following which the dNTPs, primers, and template DNA were added and the PCR cycle commenced.
  • DNA sequencing and Restriction Analysis was firstly its known high cutting frequency within the 16s rDNA genomic template, which was evident following sequence analysis of the amplified fragments from the fourteen bacterial species, and secondly the ability to heat inactivate the enzyme prior to addition of template DNA.
  • Amplified DNA from PCR reactions were treated as described for the fungal samples in Example 1, with the exception that the primers used were SEQ ID NO: 22 and SEQ ID NO: 23. A search was made for a total of 268 restriction enzyme recognition sites. Restriction enzyme analysis
  • restriction enzyme cocktail (5 units each enzyme) was added directly to approximately 1 ⁇ g of DNA/PCR product in PCR buffer which had been adjusted to contain a final concentration of 2mM Tris HC1 (pH8.3). Differentiation into Gram positive and Gram negative species was achieved using restriction enzymes SnaBI and Stuli ' n combination. The reactions were incubated at 37°C for two hours and halted either by freezing or addition of gel loading buffer containing 1 OmM EDTA.
  • restriction enzyme cocktail (5 units each enzyme) was added to approximately 1 ⁇ g of DNA PCR product in PCR buffer which had been adjusted to contain 1 OOmM NaCl, ImM DTT, and a final concentration of 7mM MgCl .
  • a restriction enzyme cocktail containing the following nine restriction endonucleases was used to achieve speciation: AfllH, BssHII, Clal, Dral, Dralll, Hpal, Ndel, Nsil, and Sail. Restriction enzyme digests were performed at 37°C for 18 hours The reaction was halted by freezing and restriction fragments analysed on 10% TBE/polyacrylamide gels.
  • Fusarium solani or human leucocytes was used as template.
  • first round PCR product was added to the nested amplification reaction as template.
  • Control negative (no DNA) samples included water and vitreous (normal or inflamed as appropriate). Control negatives were included in each protocol and the first round negative/s were included as test samples in the nested PCR reaction. Control negative samples from both rounds of PCR were consistently negative after two rounds of amplification.
  • Control positive samples included extracted genomic DNA (lOng and lOfg) or live organisms (1-5 organisms by dilution from fresh overnight culture) in both water and vitreous. Control positive samples were consistently positive after one round (lOng extracted DNA only) and two rounds of amplification (lOfg extracted genomic DNA or 1-5 live organisms).
  • the sensitivity of the first round PCR was routinely found to be 1 Opg from dilutions of DNA from E.coli, coagulase-negative Staphylococci, S. aureus and K. pneumoniae starting from a concentration of lOng/ ⁇ l.
  • the sensitivity of the reaction was improved to lfg following nested PCR. Assuming a total DNA content of 5fg per organism, this is approximately equivalent to a sensitivity of one organism.
  • serial ten fold dilutions were made from a small 1mm colony of bacteria in water (test organisms: coagulase negative Staphylococci and E. coli).
  • PCR fragments were gene cleaned and cloned into pCRII. Subsequent PCR and sequencing was then successfully performed using primers complementary to Sp6 and T7 sites present in the pCRII vector.
  • the Sna BI and StuI recognition sites proved unique to Gram positive and negative bacterial 16s rDNA sequences, respectively, thereby allowing Sna BI to cut PCR products derived from Gram positive bacteria only, and StuI to cut PCR product derived from Gram negative bacteria. Although the patterns obtained allow differentiation into Gram positive and negative bacteria, speciation was obtained for three bacterial species; P. aeruginosa, H. i ⁇ fluenzae and P. acnes.

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Abstract

This invention relates to a method for determining the presence of ocular pathogens in a sample using the polymerase chain reaction and restriction fragment length polymorphism analysis. The method provides a simple way of accurately determining a pathogen to the species level and can detect the pathogen even if only one pathogenic organism is present in the sample.

Description

DIAGNOSIS OF OCULAR PATHOGENS Field of the Invention
This invention relates to the diagnosis of ocular pathogens. Background of the Invention
Ocular infection can result in a profound loss of vision. Infection of the cornea (keratitis) can be broadly divided into that caused by bacteria, fungi and Acanthamoeba species, whereas intraocular infection (endophthalmitis) can be either bacterial or fungal. Table 1 illustrates the most common ocular pathogens.
Table 1
The effect of infection and its consequent inflammation on the eye is considerably worse if appropriate treatment is delayed. In order to maximise the likelihood of a satisfactory visual outcome, broad spectrum intraocular and systemic antibiotics are administered as soon as samples have been collected for culture. This therapy is then adapted once the organism causing the disease is identified by culture. The clinical situation is further complicated by the fact that immune-mediated processes can appear clinically indistinguishable from infection and different organisms such as fungi and bacteria can have very similar clinical appearances. Also, conventional microbial culture techniques have a low yield of positive cultures (around 40%), from cases of presumed ocular infection, and their results take a minimum of 24 hours and a maximum of 14 days to obtain.
Fungal endophthalmitis is one example of ocular infection and accounts for 4-11% of all cases of culture proven endophthalmitis. It is usually acquired from an endogenous source but may also be secondary to intraocular surgery, corneal ulceration or trauma. The time to diagnosis from the onset of symptoms has been reported as varying from 3 days to 4 months. Fungal endophthalmitis may have a classical appearance e.g Candida albicans, but it may also present in a similar manner to chronic bacterial endophthalmitis, or it may coexist with bacterial endophthalmitis. Although C. albicans is the causative agent in the majority of cases of culture-proven fungal endophthalmitis, C. tropicalis, C. krusei, C. glabrata, and C. parapsilosis are increasingly recognised as capable of causing endophthalmitis. As some of these isolates, eg. C. glabrata, and C. krusei, demonstrate inherent resistance or reduced susceptibility to azole antifungal drugs, it is important to determine the species causing the disease at the first available opportunity. The emergence of non-albicans spp as ocular pathogens is of particular concern as very few drugs are available which, when given systemically, achieve adequate vitreous concentrations even in eyes with inflamed blood-ocular barriers. The drugs fluconazole and flucytosine achieve good penetration into the central nervous system and ocular tissues. Unfortunately, their use in clinical therapy is limited due to the resistance to flucytosine which develops very early in treatment and the occurrence of fluconazole resistant strains. Amphotericin B remains the only drug to which resistance has not been encountered in ocular isolates. This drug, however, requires intraocular injection as intravenous administration achieves adequate concentrations only in the retina and choroid but not in the vitreous. As routine clinical management of patients with fungal endophthalmitis involves one intraocular injection of amphotericin B followed by 6 weeks of oral fluconazole therapy, awareness of possible drug resistance to fluconazole is essential. Should resistance occur, the patient will not be receiving the appropriate systemic antifungal therapy to continue treatment for the diseased eye; to treat or protect the fellow eye; or to combat systemic disease. As amphotericin B levels in the vitreous cavity of a vitrectomised eye rapidly fall over a few hours, the protection for the injected eye would also soon come to an end. Bacterial endophthalmitis is a further example of a severe sight-threatening disease. Early diagnosis and appropriate treatment have been noted to be associated with a better visual outcome, but culture techniques can take between 24 hours and 14 days to confirm the presence and identity of the pathogen. Currently patients are treated with broad spectrum antibiotics as soon as possible after presentation and this treatment is then later customised once results of culture are available. Many cultures, however, prove to be negative, and although this may reflect the purely inflammatory nature of the disease in some cases, it may also be a reflection of the presence of only a small number of bacteria in the sample, some of which may be non-viable following antibiotic treatment and attack from the host's immune defences. This small number may also be a reflection of the difficulties in effective sampling as bacteria can reside in biofilms on intraocular lenses (IOL).
In recent years, polymerase chain reaction (PCR) technology has shown great potential in detecting and identifying low copy numbers ofDNAin clinical samples. It also holds great promise in allowing identification of small numbers of organisms in small sample volumes, as is usually the case in ocular samples from patients with endophthalmitis.
However, previous use of PCR to diagnose infection has been too specific, amplifying one or a few particular species, or has been insensitive and not applicable to the small numbers of organisms encountered in samples from patients with keratitis or endophthalmitis. Summary of the Invention
The present invention relates to a method for the rapid diagnosis of the presence or absence of ocular infection, and the identification of pathogens to the species level. In particular, the present invention discloses a method for determining the presence of ocular pathogens in a sample, comprising the steps of:
(i) subjecting the sample to conditions under which nucleic acids are extracted;
(ii) amplifying the nucleic acids using a set of bacterial, fungal or Acanthamoeba -specific oligonucleotide primers; (iii) amplifying the product of step (ii) using nested or semi-nested bacterial, fungal or Acanthamoeba-specific oligonucleotide primers; and (iv) detecting any second amplified product.
The method is very sensitive and a diagnosis can be made for fungi even if only one gene copy is present and for bacteria if only one organism is present. Furthermore, the method significantly reduces the time taken to make a diagnosis. The invention is based on the finding that nested or semi-nested oligonucleotide primers can be used to improve sensitivity in the amplification of pathogen DNA from an ocular sample. Two amplification steps are required: the first requires oligonucleotide primers having broad specificity for ocular pathogens; the second uses nested or semi- nested primers to re-amplify the first step products and to provide genus-specific and in some instances species-specific information. The further use of restriction enzymes may provide species-specific information in the majority of cases. Detailed Description of the Invention
To ensure that all potential pathogens are detected, the first amplification step uses a pair of oligonucleotide primers designed to amplify highly conserved gene sequences. The primer pairs for bacterial pathogens, fungal pathogens and Acanthamoeba species may initially be used together to indicate whether or not an ocular pathogen is present in a sample.
The products from this first amplification step are then re-amplified using nested or semi-nested oligonucleotide primers. It is preferable that these primers also amplify highly conserved gene sequences of bacteria, fungi and Acanthamoeba and are genus- specific rather than species-specific. It is therefore possible to identify broadly the organisms most likely causing infection, i.e. either bacterial, fungal or Acanthamoeba species, immediately following the second amplification step.
If detection to the species level is required, the products from either the first or second step can be treated with a cocktail of restriction enzymes. Each species will have a particular restriction enzyme map. By using various combinations of restriction enzymes, it may be possible to identify the different species present on the basis of their individual restriction enzyme maps.
The following procedures illustrate the steps that may be taken to isolate the pathogen DNA from a patient with suspected endophthalmitis or keratitis. They are applicable to each of bacterial, fungal and Acanthamoeba pathogens. Alternative procedures will be apparent to those skilled in the art. Procedure for endophthalmitis cases
Intraocular sampling is routinely undertaken on all patients with suspected endophthalmitis. The extraocular environment was sterilised with 5% Povidone Iodine solution prior to surgery. Approximately 100-200 μl of aqueous fluid was withdrawn using a 27G (0.33mm) needle via a limbal paracentesis. Vitreous samples (200-400 μl) were taken as a biopsy through the pars plana. Ocular samples were examined by Gram and Giemsa staining, and cultured on blood agar, Sabauraud's agar, in Brain Heart Infusion (BHI) and Robertson's Cooked Meat Broth (CMB). Cultures were incubated under both aerobic and anaerobic conditions. All cultures were maintained for 14 days. Isolates were maintained at -70°. Procedure for keratitis cases
Corneal scrapes were performed at the slit lamp under topical anaesthesia. Sterile 27G needles were used to scrape off sections of corneal epithelium and anterior stroma in the region of the leading edge of the corneal ulcer. Samples were placed on a slide for Gram and Giemsa staining and also plated immediately on Blood and Sabauraud's non- nutrient agar. Plates were incubated under aerobic conditions at 25°C, 30°C or 37°C as appropriate.. Normal Vitreous
Vitreous was collected by sterile technique at the time of vitrectomy during planned surgical procedures in patients with no evidence of intraocular infection or inflammation or medical history of uveitis and/or diabetes mellitus. Samples of normal vitreous were aliquoted in a sterile manner and stored at -20 °C. DNA extraction 1) Fungi Method 1: Full extraction procedure
DNA was extracted by a method which was modified from that described by Buchman et al, Surgery (1990) 108:338-347. DNA extraction tubes were prepared as follows: each tube contained 5 μ 1 of neat vitreous, 100 μl of 0.05 mol/1 Tris pH 7.5, 0.01 mol/1 EDTA, 0.028 mol/1 β-mercaptoethanol containing 0.3mg/ml zymolase and 5 μl of the required dilution of C. albicans in water. Samples were incubated for 30 minutes at 37°C, followed by addition of 0.1% SDS and 15 μg/ml proteinase K, and incubation continued for a further 5 minutes. The mixture was subsequently heated to 95 °C for 5 minutes and cooled on ice for 15 minutes. Samples were extracted with phenol-chloroform-isoamyl alcohol (25:24:1, pH 8.0), chloroform and precipitated with 2.5 volumes of ethanol in the presence of 300mM sodium acetate. The DNA was pelleted at 14,000g for 20 minutes, washed with ice cold 80% ethanol, air dried at 65 °C for 10 minutes and resuspended in 25 μl of sterile TE buffer (lOmM Tris, ImM EDTA, pH8.0). Method 2: Boil and PCR The DNA extraction tubes were prepared as follows: each tube contained 5 μl of neat vitreous and 5 μl of the required dilution of C. albicans in water. Samples were incubated at 95 °C for 20 minutes and subsequently the entire volume was used in PCR reactions. 2) Bacteria Method 1: Full extraction procedure
Previous work in this laboratory has successfully used glass beads and a bead beater apparatus (Stratech Scientific) to effectively release DNA from bacterial cells in suspension (Hykin et al, J. Med. Micro. (1994) 40:408-415). A 2mm colony of bacteria was diluted into 300μl sterile PBS solution, with 50 μg/ml proteinase-K and approximately 0.5g glass beads. This mixture was beaten on the bead beater on full power for 10 seconds prior to the addition of phenol (pH 8.0). Samples were vortexed, and centrifuged for 2 minutes at 14,000g in a microfuge and the aqueous phase removed and re-extracted with phenol/chloroform/isoamyl alcohol (25:24: 1), and chloroform. DNA was then precipitated with 1/10 volume 3M sodium acetate (pH 5.3) and 1 volume isopropanol at -20 °C. The DNA was pelleted at 14,000g, for 20 minutes, washed with ice cold 80% ethanol, air dried at 65 °C for 10 minutes and resuspended in 25 μl of sterile TE buffer (lOmM Tris, 1 mM EDTA, pH 8.0). Method 2: Boil and PCR:
Bacteria were suspended in the PCR mix and an initial denaturation step of 95° for 5 minutes was added to the PCR cycle. 3) Acanthamoeba spp.
Method 1: Full extraction procedure
The method described by Ledee et al, Investigative Ophthalmology and Visual Science, (1996) 37:544-550 will be followed. Acanthamoeba were lysed in 500μl of UNSET buffer (8 M urea,, 0.15M NaCl, 2% SDS, O.OOIM EDTA, 0.1 M Tris pH 7.5). The lysate was phenol-chloroform extracted and the aqueous layer removed and made 0.3M with NaCl. Nucleic acids were then precipitated with two volumes of ethanol. Method 2: Boil and PCR:
A simplified DNA extraction is reported by Lai et al, Molecular and Cellular Probes, (1994) 8:81-89. Incubation of Acanthamoeba spp in modified PCR buffer for 1 hour and boiling for 10 minutes was sufficient to release DNA from cells. PCR assay Design and optimisation of primers.
Examples of primers used to amplify a highly conserved gene sequence in a pathogen, appear in Table 2. For bacterial pathogens, a suitably conserved gene is the 16S rDNA gene. For yeast pathogens, primers to the cytochrome P450 L^ demethylase gene can be used. The 18S ribosomal gene may be used for fungal pathogens. Further embodiments of the invention are as described in the sub-claims. Other suitably conserved genes that may be used as targets for amplifying yeast, bacterial ox Acanthamoeba DNA, will be apparent to those skilled in the art.
Replitherm Taq (Cambio) DNA polymerase was used for bacterial PCR, whereas, Amplitaq (Perkin-Elmer) DNA polymerase was used for yeast and Acanthamoeba PCR.
Table 2
Optimisation for annealing temperature, and magnesium concentration can be carried out to find the primer sets which produce the highest product yield and greatest sensitivity whilst retaining high specificity.
The following examples illustrate the invention with respect to the identification of fungal pathogens (Example 1) and bacterial pathogens (Example 2). Example 1
Seven novel primers, based on the Cytochrome P450 Lj At demethylase gene were used to detect Candida spp and were optimised and formed two nested pairs. Cytochrome P450 Lj A, demethylase was chosen for PCR amplification because its product catalyses an essential step in the conversion of lanosterol to ergosterol. Ergosterol is unique to fungi and yeast cell walls and is not detected in human cells or common bacterial pathogens. Detection and identification of fungi using this gene may also allow more detailed analysis of the active site region by DNA sequencing of resistant and susceptible strains and ultimately the design of primers to detect the presence of specific mutations. Optimal PCR conditions
Both first round and second round reactions contained 100 μM each dNTP (Pharmacia), 200 μM of each primer, 2.5mM magnesium chloride, 1 unit DNA polymerase lOOmM Tris-HCl ( pH 8.3) 500mM KC1; 15mM MgCl2; 0.001%w/v gelatin, in a 50 μl reaction. Cycling conditions were identical for both rounds except for the annealing temperature which was found to be optimal at 51.5 ° C for the first round (primers SEQ ID NO: 7 & 8), 56°C for nested primers I (SEQ ID NO: 9 & 10) and 58 °C for semi-nested primers II (SEQ ID NO: 9 & 8). PCR cycling was performed at 95 °C for 10 minutes for one cycle, followed by 30 cycles of 94 ° C for 1 minute, annealing temperature as above for 1 minute, and extension at 72°C for 1 minute. The final cycle was followed by 10 minutes at 72 °C.
Electrophoresis and imaging
PCR products were resolved on a 1-2 % agarose/TBE gel according to the size of fragment expected and visualised using ethidium bromide under UV illumination.
DNA sequencing and Restriction maps
Amplified DNA from PCR reactions were purified on agarose/TBE gels, excised, and recovered into solution. PCR fragments were directly cycle sequenced in both directions (using primers SEQ ID NO: 7 & 9) on an ABI prism automated DNA sequencer (model 377 version 2.1.1). Cycle sequencing was repeated 3 times for each product using the same DNA and PCR protocol but different PCR reactions. Sequences were aligned and scanned both manually and with the aid of GCG and REBASE programs provided by the HGMP computer centre. A search was made for a total of 204 restriction endonuclease sites. RFLP analysis
Following PCR amplification, products were subjected to phenol: chloroform:isoamyl alcohol (25:24: 1, pH 8.0) followed by chloroform extraction, and ethanol precipitation. Precipitated DNA was resuspended in ddH20 prior to digestion with restriction endonuclease enzymes. Restriction enzyme combinations were used which yielded fragments that allowed easy identification of species following separation of digested DNA on polyacrylamide/TBE gels.
Restriction enzyme digests were performed at 37°C for 2 hours in buffer containing 6mM Tris -HC1, 6mM Mg Cl2, 150 mM NaCl at pH7.9 and 10 units of each enzyme. The resulting fragments were subsequently resolved on 4-8% polyacrylamide gels and visualised under UV illumination following staining with ethidium bromide.
Specificity of the fungal specific primers
Specificity of the outer primer pair SEQ ID NO: 7 & 8 and semi-nested primers
II (SEQ ID NO: 9 & 8) was demonstrated by the amplification of a single 1.0 kb and 500bp fragment, respectively, from all Candida spp tested except C. guillermondii.
Specificity of the nested primer pair I (SEQ ID NO: 9 & 10) was demonstrated by the amplification of a single 270bp fragment from Candida albicans only. No amplification product was obtained using genomic DNA isolated from Aspergillusβimigatus, Fusarium solani, human leucocytes or 13 species of bacteria. The specificity of all the primer combinations is summarised in Table 3, where CA = C. albicans, CT = C. tropicalis, CP = C. parapsilosis, CG = C. glabrata, Cpell = C. pelliculosa, CK = C. krusei and Cgul = C. guillermondii.
Table 3
Sensitivity of PCR reactions using dilution series of extracted DNA
The sensitivity of the first round PCR was routinely found to be 30-300 pg from dilutions of DNA from C. albicans starting from a concentration of lOng/μl. Assuming a total DNA content of 37fg per organism, and two copies of the gene per diploid genome this is equivalent to 800-8000 organisms. The second round of PCR markedly improves this sensitivity to 10 fg which is equivalent to less than one gene copy. As lOfg is less than the concentration of DNA in one organism, we proceeded to PCR amplify 1 Ofg of C. albicans genomic DNA twenty times expecting a positive result one time out of three. A fresh DNA dilution series was prepared using 1.5 μg of C. albicans genomic DNA (a concentration at which spectrophotometric DNA concentration measurements are reliable). Two rounds of PCR amplification using SEQ ID NO: 7 & 8 followed by SEQ ID NO: 9 & 10, yielded a positive result from 13 of the 20 tubes (65%). These observed results approximate that which would be expected if one assumes one copy of the gene in every 18.5 fg of genomic DNA would yield a positive result in 54% of the tubes containing 1 Ofg of DNA.
Sensitivity of the PCR with dilution series of live organisms in vitreous
The results obtained using extraction methods one and two were identical. Using method 2, and after first round PCR (with primers SEQ ID NO: 7 & 8), only the control positive was found to have been amplified to such an extent that the l.Okb product could be detected by ethidium bromide staining of agarose gels after electrophoresis. Subsequent nested PCR reactions demonstrated clear amplification products of the predicted size from all dilutions. Control negatives were included in each protocol and the first round negatives were included as a test sample in the nested PCR reaction. Control negative samples from both rounds of PCR were consistently negative after two rounds of amplification.
Microscopic analysis of dilution series
Microscopic analysis of the numbers of organisms in the most dilute tubes revealed a concordance of 100% between the number of organisms seen in 5 μl of each dilution under the microscope and the number of organisms which grew in culture. This could only be carried out in high accuracy in the more dilute tubes. Microscopy also identified the presence of large clumps of cells (in the more concentrated tubes) which would indicate poor mixing, revealing the low reliability of pipetting as a mixing process and the low likelihood of achieving similar numbers of organisms in different aliquots of the same dilution fluid. However, the lowest dilution at which organisms could be seen under the microscope, was also the dilution at which the same small number of organisms grew in culture, indicating a high concordance between microscopy and culture in these most dilute samples.
In the dilutions in which culture and microscopy were both negative for organisms but PCR was positive, a variable amount of debris was seen, some of which could possibly have been cellular in origin. Since DNAse treatment of the original colony was performed to destroy all free DNA prior to the preparation of the dilution series, these results would indicate the break up of cells during handling, and the release of DNA into the mixture which may have been PCR amplified but not detected as viable cells by microscopy and/or by culture. DNA amplification from spiked vitreous The detection of first round PCR product was successful by ethidium bromide staining of agarose gels in the presence of vitreous if the concentration of vitreous did not exceed 20% of the total reaction volume. RFLP analysis
The combination of Atw26I + Spel successfully differentiated between the 1.Okb PCR amplified products from all 6 yeasts. The use of Eco RJ+ BstXl+ Xba 1+ Nde 1 in combination successfully differentiated between the 500bp PCR amplified products from all yeasts. The 500 bp fragment amplified from C. parapsilosis genomic DNA remained undigested. DNA sequencing The full sequences have been published: C. albicans (Ref.1), C. tropicalis ( Ref.2), and S. cerevisiae (Ref.3) and partial sequences from C. krusei (Ref.4) , C. glabrata (Ref.4), C. parapsilosis (Ref.5) , C. kefyr (Ref.5), and C. guillermondii (Ref.5). Genbank accession numbers are AFO 19902 and AFO 19903 for C. parapsilosis and C. pelliculosa respectively. Example 2 PCR-RFLP was performed on eight Gram positive and six Gram negative organisms known to be causative agents of endophthalmitis. Two pairs of oligonucleotide primers based on the 16s rDNA gene were used to PCR amplify a 1.2 kb and a 1.0 kb fragment of bacterial genomic DNA. Restriction fragment length polymorphisms within the PCR product were identified following sequence analysis of the amplified products and subsequently used to speciate the organisms following restriction analysis.
16s rDNA genes are ubiquitous and are present as multiple copies within bacterial genomes. Optimisation for annealing temperature, and magnesium ion concentration were undertaken in an attempt to find the experimental conditions which produced the highest product yield and greatest sensitivity whilst retaining high specificity. Optimal PCR conditions
PCR reactions contained 20mM Tris-HCl (pH8.3), 100 mM KC1, 0.01% Tween 20, 0.1% NP-40, lOOμM each deoxyribonucleotide ('dNTP', Pharmacia Biotech, St Albans, UK), 0.3 μM of each primer, 1.5mM magnesium chloride, 3 units Replitherm Taq DNA polymerase ('Taq', Cambio Ltd. Cambridge, UK), in a 25 μl reaction. Both first round and nested PCR cycles commenced with 5 minutes at 95 °C. First round PCR with outer primers SEQ ID NO: 22 & 23, was performed by cycling at 94 °C for 10 seconds, 56.2 °C annealing for 10 seconds, and 72°C for 15 seconds for 30 cycles. Cycling with nested primers SEQ ID NO: 24 & 25 was performed at 94°C for 7 seconds, 64 °C annealing for 7 seconds, and 72°C for 10 seconds for 30 cycles.
The Taq DNA polymerase used in this study is one of a number of commercially available Taq polymerases with known low levels of DNA contamination. Although this level of contamination is insufficient to give a detectable amplification product after just one round of PCR, it is easily detected following nested amplification. Therefore, in this study, prior to first round PCR amplification, the Replitherm Taq was treated with Alul restriction endonuclease (ratio of 3 : 1 units of Taq: Alul, Promega UK Ltd., Southampton, UK), which recognises a specific nucleotide sequence AGCT. The criteria for choosing this restriction enzyme was firstly its known high cutting frequency within the 16s rDNA genomic template, which was evident following sequence analysis of the amplified fragments from the fourteen bacterial species, and secondly the ability to heat inactivate the enzyme prior to addition of template DNA. Prior to PCR amplification the water, PCR buffer, magnesium and Taq components were mixed and incubated at 37°C with Alul for 30 minutes. The restriction enzyme was subsequently inactivated by incubation at 95 °C for two minutes, following which the dNTPs, primers, and template DNA were added and the PCR cycle commenced. DNA sequencing and Restriction Analysis
Amplified DNA from PCR reactions were treated as described for the fungal samples in Example 1, with the exception that the primers used were SEQ ID NO: 22 and SEQ ID NO: 23. A search was made for a total of 268 restriction enzyme recognition sites. Restriction enzyme analysis
Following PCR amplification, the concentration of PCR product was estimated using ethidium bromide staining of agarose/ TBE gels. Restriction enzyme combinations were used which yielded fragments which allowed easy identification of species following separation of digested DNA on polyacrylamide/TBE gels. Two restriction enzyme cocktails were developed, one to differentiate organisms into Gram positive and Gram negative species (protocol A) and a second cocktail to directly speciate organisms (protocol B). RFLP protocol A
The restriction enzyme cocktail (5 units each enzyme) was added directly to approximately 1 μg of DNA/PCR product in PCR buffer which had been adjusted to contain a final concentration of 2mM Tris HC1 (pH8.3). Differentiation into Gram positive and Gram negative species was achieved using restriction enzymes SnaBI and Stuli'n combination. The reactions were incubated at 37°C for two hours and halted either by freezing or addition of gel loading buffer containing 1 OmM EDTA. RFLP protocol B
The restriction enzyme cocktail (5 units each enzyme) was added to approximately 1 μg of DNA PCR product in PCR buffer which had been adjusted to contain 1 OOmM NaCl, ImM DTT, and a final concentration of 7mM MgCl . A restriction enzyme cocktail containing the following nine restriction endonucleases was used to achieve speciation: AfllH, BssHII, Clal, Dral, Dralll, Hpal, Ndel, Nsil, and Sail. Restriction enzyme digests were performed at 37°C for 18 hours The reaction was halted by freezing and restriction fragments analysed on 10% TBE/polyacrylamide gels.
Specificity
Using primers SEQ ED NO: 22 & 23 a 1.2 kb product was obtained from all strains of all species of bacteria tested. When P.acnes genomic DNA was used as template, a 600 bp was also obtained and when P. aeruginosa genomic DNA was used as template a faint
200bp fragment was usually visible.
Using primers SEQ LD NO: 24 and SEQ ID NO: 25, a single 1.0 kb amplification product was obtained using genomic DNA from all bacteria tested. No amplification product was obtained when genomic DNA from Candida albicans, Aspergillusfumigatus,
Fusarium solani, or human leucocytes was used as template.
Nested PCR protocol and controls
In each case, lμl of first round PCR product was added to the nested amplification reaction as template. Control negative (no DNA) samples included water and vitreous (normal or inflamed as appropriate). Control negatives were included in each protocol and the first round negative/s were included as test samples in the nested PCR reaction. Control negative samples from both rounds of PCR were consistently negative after two rounds of amplification.
Control positive samples included extracted genomic DNA (lOng and lOfg) or live organisms (1-5 organisms by dilution from fresh overnight culture) in both water and vitreous. Control positive samples were consistently positive after one round (lOng extracted DNA only) and two rounds of amplification (lOfg extracted genomic DNA or 1-5 live organisms).
Sensitivity of PCR reactions The sensitivity of the first round PCR was routinely found to be 1 Opg from dilutions of DNA from E.coli, coagulase-negative Staphylococci, S. aureus and K. pneumoniae starting from a concentration of lOng/μl. The sensitivity of the reaction was improved to lfg following nested PCR. Assuming a total DNA content of 5fg per organism, this is approximately equivalent to a sensitivity of one organism. To assess the sensitivity of detection with live organisms, serial ten fold dilutions were made from a small 1mm colony of bacteria in water (test organisms: coagulase negative Staphylococci and E. coli). Equal aliquots were immediately cultured and amplified in PCR reactions. The sensitivity of detection after one round of PCR was approximately 600 organisms for both species. This sensitivity was improved to one organism after a second round of PCR. The preparations which were the most dilute (10"7 - 10'12 dilutions) had aliquots which were both culture and PCR positive, both culture and PCR negative, and occasionally PCR positive but culture negative but never culture positive and PCR negative. DNA sequencing
Each PCR product was sequenced three times. Comparison of the DNA sequences obtained made with the full and partial sequences of the genes already available in Genebank demonstrated that they were derived from the 16s rDNA gene (sequences 97-100% identical). The sequences obtained were submitted to Genebank and have been awarded the following accession numbers: Streptococcus pyogenes: AFO76028, Streptococcus viridans: AFO76036, Streptococcus faecalis: AFO76027, Propionibacterium acnes: AFO76032, Staphylococcus aureus: AFO76030, Streptococcus pneumoniae: AFO76029, Bacillus cereus: AFO76031, Escherichia coli: AFO76037, Serratia marcescens: AFO76038, Haemophilus inβuenzae: AFO76035, Klebsiella pneumoniae: AFO76033, Proteus mirabilis: AFO76034, and Pseudomonas aeruginosa: AFO76039.
Strain testing A set of control patterns was obtained from all fourteen bacterial species.
Subsequently, PCR-RFLP and sequence analysis was performed on all clinical isolates to ensure the reproducibility of the procedure. All clinical isolates of Streptococci, S. aureus and each of the Enterobacteriacae, results of PCR, RFLP and sequencing were in agreement. RFLP analysis of coagulase-negative Staphylococcal species yielded three patterns. Two of these were still identified as coagulase-negative Staphylococci by a masked observer but a third pattern was found to be un-identifiable. Sequence analysis of clinical isolates of coagulase-negative Staphylococci yielded 98% base identity in each case with several of the following Staphylococcal species which belong to this group: S. epidermidis, S. caprae, S. capitis, S. warneri, S. lugdunensis and S. pasteurii. These sequences were also found to be 97% identical to that of S. aureus. The converse was also found to be true of S. aureus sequences. These new RFLP patterns have been added to the "control patterns" to aid future identification of these species.
Cloning of PCR products
Sequencing direct from PCR product did not yield adequate sequencing data for the following bacteria: P.mirabilis, H. iηfluenzae, S. viridans and P. acnes. PCR fragments were gene cleaned and cloned into pCRII. Subsequent PCR and sequencing was then successfully performed using primers complementary to Sp6 and T7 sites present in the pCRII vector.
RFLP analysis of amplified PCR products from 14 bacterial spp RFLP analysis differentiates bacterial spp into Gram positive and Gram negative The use of just two enzymes (Sna BI and StuI) successfully differentiated between
Gram positive and Gram negative organisms. The Sna BI and StuI recognition sites proved unique to Gram positive and negative bacterial 16s rDNA sequences, respectively, thereby allowing Sna BI to cut PCR products derived from Gram positive bacteria only, and StuI to cut PCR product derived from Gram negative bacteria. Although the patterns obtained allow differentiation into Gram positive and negative bacteria, speciation was obtained for three bacterial species; P. aeruginosa, H. iηfluenzae and P. acnes.
RFLP analysis differentiates bacterial spp
The combination of AflHI, BssHII, Clal, Dral, Dralll, Hpal, Ndel, Nsil, and Sα/7 successfully differentiated between the PCR amplified products from 13 of 14 bacterial species. E. coli and S. marcescens yielded identical RFLP patterns and therefore could not be interdifferentiated.
References:
1 Lai MH, Kirsch DR. Nucleotide sequence of cytochrome P450 LI Al (lanosterol
14 alpha-demethylase) from Candida albicans. Nucleic Acids Res. 1989; 17: 804
2. Chen C, Kalb VF, Turi TG, Loper JC. Primary structure of the cytochrome P450 lanosterol 14 alpha-demethylase gene from Candida tropicalis. DNA. 1988; 7:
617-26
3. Kalb VF, Woods CW, Turi TG, Dey CR, Sutter TR, Loper-JC. Primary structure of the P450 lanosterol demethylase gene from Saccharomyces cerevisiae. DNA. 1987; 6: 529-37 4. Burgener-Kairuz P, Zuber JP, Jaunin P, Buchman TG, Bille J, Rossier M. Rapid detection and identification of Candida albicans and Candida glabrata in Clinical Specimens by Species-Specific Nested PCR Amplification of a Cytochrome P450 lanosterol -α- demethylase (L1A1) Gene Fragment. J Clin Microbiol. 1994;32: 1902-1907 5. Morace G, Sanguinetti M, Posteraro B, Lo-Cascio G, Fadda-G. Identification of various medically important Candida species in clinical specimens by PCR- restriction enzyme analysis. J Clin Microbiol. 1997; 35: 667-72.

Claims

I. A method for determining the presence of ocular pathogens in a sample, comprising the steps of:
(i) subjecting the sample to conditions under which DNA is extracted; (ii) amplifying the DNA using a set of bacterial, fungal or Acanthamoeba- specific oligonucleotide primers; (iii) amplifying the product of step (ii) using nested or semi-nested bacterial, fungal or Acanthamoe ba-specific oligonucleotide primers; and (iv) detecting any second amplified product.
2. A method according to claim 1, wherein the pathogen is fungal, bacterial or protozoan.
3. A method according to claim 2, wherein the pathogen is Candida species.
4. A method according to claim 2, wherein the pathogen is a gram +ve bacterium.
5. A method according to claim 2, wherein the pathogen is a gram -ve bacterium.
6. A method according to claim 2, wherein the pathogen is Acanthamoeba species.
7. A method according to claim 2, wherein the pathogen is a cause of keratitis.
8. A method according to claim 2, wherein the pathogen is a cause of endophthalmitis.
9. A method according to claim 2, wherein the oligonucleotide primers of step (ii) are selected from those defined herein as SEQ ID NO: 1 to 6, or functional equivalents thereof.
10 . A method according to claim 3, wherein the oligonucleotide primers are complementary to nucleotide sequences on the cytochrome P450 L1 A, demethylase gene.
I I. A method according to claim 10, wherein the oligonucleotide primers of step (ii) are those defined herein as SEQ ID NO: 7 and SEQ ID NO: 8, or functional equivalents thereof, and the oligonucleotide primer of step (iii) are selected from any of those defined herein as SEQ ID NO: 9 to 18, or functional equivalents thereof.
12. A method according to claim 4 or claim 5, wherein the oligonucleotide primers are complementary to nucleotide sequences on the bacterial 16s rDNA gene.
13. A method according to claim 12, wherein the oligonucleotide primers of step (ii) are those defined herein as SEQ ID NO: 22 and 23, or functional equivalents thereof, and the oligonucleotide primers of step (iii) are selected from any of those defined herein as SEQ ID NO: 24 to 28, or functional equivalents thereof.
14. A method according to claim 6, wherein the oligonucleotide primers of step (ii) are selected from any of those defined herein as SEQ ID NO: 29 to 32, or functional equivalents thereof, and the oligonucleotide primers of step (iii) are those defined herein as SEQ ID NO: 33 and 34, or functional equivalents thereof.
15. A method according to any preceding claim, wherein step (i) comprises incubating the sample at a temperature above 90┬░C.
16. A method according to any preceding claim, wherein the amplification reaction has an annealing temperature of at least 51.5 ┬░ C.
17. A method according to any preceding claim, wherein either or each amplified product is treated with one or more restriction enzymes.
18. A method according to any preceding claim, wherein the sample is from the cornea or intraocular fluids.
19. The use of nested oligonucleotide primers capable of hybridising to the cytochrome P450 L,Aj demethylase gene, for detecting Candida species in samples from the cornea or intraocular fluids.
20. The use according to claim 19, wherein the primers hybridise to the demethylase gene between nucleotides 157-361 and 382-1560 of the said gene.
21. The use according to claim 19 or claim 20, wherein the primers are as defined in claim 10, or the complement thereof.
22. An oligonucleotide primer capable of high stringency hybridisation to a genus- specific sequence of the Cytochrome P450 L,A, demethylase gene between nucleotide numbers 157-361 and 382-1560.
23. An oligonucleotide primer according to claim 22, wherein the primers are as defined in claim 10, or the complement thereof.
24. An oligonucleotide primer capable of high stringency hybridisation to a genus- specific sequence of the bacterial 16S rDNA gene.
25. An oligonucleotide primer according to claim 24, wherein the primers are as defined in claim 13, or the complement thereof.
26. A kit for the detection of ocular pathogens, comprising separate packages of bacterial, fiingal or Acanthamoeba-specific oligonucleotide primers and of nested or semi- nested bacterial, fungal or Acanthamoeba-specific oligonucleotide primers.
27. A kit according to claim 26, additionally comprising restriction enzymes.
28. A kit according to claim 26 or claim 27, wherein the oligonucleotide primers are as defined in any of claims 19 to 25.
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