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WO2003023062A2 - Method of diagnosis for c.trachomatis infection using dna amplification assay on liquid cytology samples - Google Patents

Method of diagnosis for c.trachomatis infection using dna amplification assay on liquid cytology samples

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Publication number
WO2003023062A2
WO2003023062A2 PCT/GB2002/004171 GB0204171W WO03023062A2 WO 2003023062 A2 WO2003023062 A2 WO 2003023062A2 GB 0204171 W GB0204171 W GB 0204171W WO 03023062 A2 WO03023062 A2 WO 03023062A2
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WO
WIPO (PCT)
Prior art keywords
sample
cells
dna amplification
amplification assay
pellet
Prior art date
Application number
PCT/GB2002/004171
Other languages
French (fr)
Other versions
WO2003023062A3 (en
Inventor
Rukhsana Ahsan
Original Assignee
Sonic Clinical Institute Pty Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB0122180A external-priority patent/GB0122180D0/en
Application filed by Sonic Clinical Institute Pty Limited filed Critical Sonic Clinical Institute Pty Limited
Publication of WO2003023062A2 publication Critical patent/WO2003023062A2/en
Publication of WO2003023062A3 publication Critical patent/WO2003023062A3/en

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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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • the present invention relates to methods of diagnosis for C. trachomatis infection in a patient using DNA amplification assays to identify the presence of the infectious agent in a liquid cytology sample.
  • the sampling device is rinsed into a collection vial containing a transport medium of buffered methanol,
  • PreservCyt ® The sample vial is then sent to a laboratory for analysis where slides are prepared from the liquid based sample.
  • the improved method provides for the preparation of slides for analysis by automated means.
  • the analysis of slides prepared by this method for the presence of atypical cells, cervical cancer or its precursor lesions is now the approved means for diagnosis in the USA by the Food & Drug Administration (FDA).
  • FDA Food & Drug Administration
  • the ThinPrep ® Pap TestTM has also now been licensed for use in the diagnosis of human papillomavirus (HPV) by Digene Corporation (www . di gene . com) .
  • HPV test uses the ThinPrep ® sample vial produced by Cytyc Corporation containing PreservCyt ® transport medium for the ThinPrep ® Pap TestTM and analysis of the presence of HPV is carried out using the Digene Hybrid Capture HPV DNA Assay. This development was also important as nearly all cases of cervical cancer are positive for the presence of HPV. There are, though, other important diseases which if not diagnosed and treated early enough can lead to infertility problems for women.
  • the sample for analysis is prepared according to a method of the present invention which comprises effective washing of the cellular material to remove transport or storage medium in which the sample of cervical cells was collected and which further comprises pre-dilution (or titration) of cellular material in the sample prior to PCR hybridisation and detection reaction.
  • a method for the diagnosis for the presence of Chlamydia trachomatis in an individual by means of a DNA amplification assay comprising washing and pre-dilution of a sample of cells with a wash buffer that comprises a neutral buffer solution and a detergent, and optionally a halide salt of an alkali metal or an alkaline earth metal, in which the sample of cells is present in a sample collection medium after collection from the individual, wherein the washing and pre- dilution is carried out prior to lysis of the cells for the DNA amplification assay.
  • the presence of DNA from C. trachomatis in a sample as indicated by a valid, positive result from the DNA amplification assay may indicate the presence of a systemic infection by the organism in the individual being tested, or it may indicate recent introduction of the organism to the individual through close personal contact with another infected individual.
  • the DNA amplification assay may be the Polymerase Chain Reaction (PCR).
  • PCR Polymerase Chain Reaction
  • One suitable method based on the PCR approach is the automated COBAS AMPLICORTM system of Roche Diagnostics (F. Hoffman-La Roche Ltd. - www.roche.com).
  • the DNA of C. trachomatis to be amplified is extracted from chlamydial reticulate bodies obtained from cells sampled from an individual patient.
  • Other PCR-based amplification methods can be selected as appropriate by a person skilled in the art.
  • the cells present in the sample from the individual to be assayed may be from any site (or potential site) of infection in an individuals body.
  • the cells will be of cervico-vaginal origin and will be epithelial cells.
  • the washing of the sample may be undertaken by any suitable means to remove the sample collection medium.
  • the washing of the sample may comprise addition of wash buffer to the sample present in the sample collection medium, followed by centrifugation to form a pellet.
  • An aliquot of cells in sample collection medium is washed with an equivalent aliquot of wash buffer.
  • the aliquots will be in the ratio of 1 : 1 and this can be varied as required up to an addition of wash buffer in excess in order to complete full washing of the cells in the sample to remove sample collection medium.
  • the wash buffer may comprise any suitable neutral buffer and detergent, optionally including saline, that can preserve the integrity of the cells in the sample whilst permitting washing out of the sample collection medium.
  • a buffer can be defined as a substance which when added to a solution resists a change in hydrogen ion concentration on addition of acid or alkali (Grant & hackh's Chemical Dictionary, McGraw-Hill Book Company, 5 th edition (1987)).
  • a neutral pH is typically about pH 7.0, however the effective pH range of the buffer solutions of the present invention may be in the range of from pH6.9 to pH8.2, suitably pH7.4 to pH7.8, where a convenient working pH may be pH7.6.
  • Suitable buffers include but are not limited to buffer solutions prepared from tris(hydroxymethyl)aminomethane (or tris base or Trizma ® base), or its hydrochloride equivalent (Tris HC1 or Trizma ® HC1). Tris buffered saline tablets may also be used to prepare alternative versions of the wash buffer that contain saline in the form of sodium chloride.
  • Hepbs N-[2-Hydroxyethyl]piperazine-N'-[4-butanesulfonic acid] which is a homolog of Hepes and Epps with a higher pKa
  • Taps N-tris[Hydroxymethyl]methyl-3- aminopropane sulfonic acid or [2-Hydroxy-l,l-bis(hydroxymethyl)ethyl]amino)-l-propane sulfonic acid).
  • the molarity of the buffer may be in the range of from lOmmol/L to 500mmol/L, preferably from 25mmol/L to 150mmol/L, suitably from 50mmol/L to lOOmmol/L.
  • the detergent substance added to the buffer solution may be any convenient detergent that can preserve the integrity of the cells in the sample whilst permitting washing out of the sample collection medium.
  • the detergent is a non-ionic detergent.
  • the detergent may be present in the range of from 0.05% to 0.25%, preferably 0.075% to 0.2%, or at 0.1%.
  • Suitable detergents include but are not limited to t-octylphenoxypolyethoxy ethanol (Triton ® ) or polyoxyethylenesorbitan monolaurate (Tween ® ).
  • Non-ionic detergents are BIGCHAP, decanoyl-N-methylglucamide, n-Decyl ⁇ -D-glucopyranoside, n-Decyl ⁇ - D-glucopyranoside, n-Decyl ⁇ -D-maltopyranoside, DeoxyBIGCHAP, n-Dodecyl ⁇ -D- glucopyranoside, n-Dodecyl ⁇ -D-maltoside, n-Dodecyl ⁇ -D-maltoside, Heptanoyl-N- methyl glucamide, n-Heptyl ⁇ -D-glucopyranoside, N-Heptyl ⁇ -D-thioglucopyranoside, n- Hexyl ⁇ -D-glucopyranoside, Igepal CA-630, 1-Monooleolyl-rac-glycerol, Nonanoyl-N- methyl glucamide, n-Non
  • t-octylphenoxypolyethoxy ethanol can be provided in different forms such as T ⁇ ton X- 100, Triton ® X-100 Reduced, Triton ® X-114, Triton ® X-405, Triton ® X-405 Reduced, Triton ® CG-110, Triton ® XL-80N, Triton ® WR-1339 or Tyloxapol.
  • Triton ® detergents are N-42, N057, N-60, X-15, X-35, X-45, X-102, X-155, X-165, X-207, X-305, X-705-70, B-1956. In many situations, Triton ® X-100 may be preferable.
  • Polyoxyethylenesorbitan monolaurate can be provided in different forms such as Span 20, Tween ® 20, Tween ® 20R, Tween ® 21, Tween ® 80, Tween ® 80R, Tween ® 80K, Tween ® 81, Tween ® 40, Tween ® 60, Tween ® 61, Tween ® 85, or Tween ® 65.
  • the polyoxyethylenesorbitan monolaurate can be Tween ® 20.
  • the halide salt of an alkali metal or an alkaline earth metal may be a fluoride, a chloride, a bromide or an iodide salt.
  • the alkali metal ion may be lithium sodium, or potassium.
  • the alkaline earth metal ion may be magnesium, or calcium.
  • the halide salt is sodium chloride.
  • the halide salt may be selected from the list of sodium fluoride, sodium chloride, potassium chlorides, magnesium chloride, or calcium chloride.
  • Sodium chloride may optionally be included in the composition of the buffer in a range of from 50mmol/L to 500mmol/L, suitably lOOmmol/L to 400mmol/L, preferably from 150mmol/L to 300mmol/L.
  • the pre-dilution or titration of the sample may be by any convenient means to dilute out cellular material.
  • the pre-dilution or titration step may comprise addition of wash buffer to the sample after washing described above.
  • the sample of cells taken from the individual patient are collected by any generally convenient means dependent upon the location of the cells to be assayed.
  • a gynaecological sample may be collected using a broom-type or a cytobrush spatula cervical sampling device.
  • the cells obtained are then rinsed into a sample collection vial containing sample collection medium.
  • the medium is buffered methanol, for example PreservCyt ® medium. Where the medium is buffered methanol, it may be suitably buffered to a neutral pH as described in detail above in realtion to the wash buffer.
  • Lysis of the cells of the individual and reticulate bodies of the C. trachomatis in the sample is necessary to retrieve DNA of the infectious agent. Lysis can be achieved by any suitable means, but typically may involve application of a lysis medium, for example a detergent.
  • the method comprises washing a sample of cells with a wash buffer comprising a neutral buffer solution of tris(hydroxymethyl) aminomethane and a detergent selected from t-octylphenoxypolyethoxy ethanol or polyoxyethylenesorbitan monolaurate, with sodium chloride present in the medium.
  • the sample of cells to be washed is present in a sample collection medium, typically buffered methanol, after collection from the individual, and wherein the washing is carried out prior to lysis of the cells for the DNA amplification assay.
  • a sample collection medium typically buffered methanol
  • a method of the present invention may comprise therefore a means for preparing a sample of cells taken from an individual for the purposes of diagnosing C. trachomatis infection, in which the sample of cells is preserved in a sample collection medium after collection from the individual, suitably the buffered methanol solution known as PreservCyt ® produced by Cytyc Corporation for the ThinPrep ® Pap TestTM.
  • a sample collection medium suitably the buffered methanol solution known as PreservCyt ® produced by Cytyc Corporation for the ThinPrep ® Pap TestTM.
  • PreservCyt ® produced by Cytyc Corporation for the ThinPrep ® Pap TestTM.
  • preservation of the sample in sample collection medium may for the purposes of preservation prior to analysis, including transportation from the site of collection to the site of analysis.
  • the sample may also be used to provide cells for the diagnosis of other medical conditions such as, for example, cervical cancer and/or human papillomavirus (HPV) infection.
  • HPV
  • the sample needs to be prepared according to a method of the present invention prior to the use of the DNA amplification assay.
  • an aliquot of wash medium is introduced to the sample; or to an aliquot from the original sample in an appropriate vessel or tube.
  • the mixture is then centrifuged in the vessel or tube to form a pellet.
  • the supernatant from pellet such as for example by aspiration using a pipette.
  • a further aliquot of wash medium is then introduced to the pellet which is resuspended in this medium by, for example, vortexing.
  • the resulting mixture is then re-centrifuged to form a pellet which is suitable for processing for the DNA amplification assay. This is achieved by adding cell lysis medium to pellet and resuspending the pellet in this medium. Finally, centrifugation of this mixture yields a supernatant that can be removed and pre-diluted (titrated) for analysis by amplification assay for C. trachomatis DNA
  • kits of parts for the diagnosis of Chlamydia trachomatis infection in a sample from an individual by means of a DNA amplification assay comprising a wash buffer that comprises a neutral buffer solution and a detergent, and optionally a halide salt of an alkali metal or an alkaline earth metal.
  • the wash buffer is prepared from Tris-HCl or Tris base (Sigma) to molarity lOOmmol/L at pH7.6.
  • the solution is completed by the addition of t-octylphenoxy polyethoxyethanol (Sigma) to 0.1% and sodium chloride to 150mmol/L.
  • Example 2 Washing of cells from collected samples Samples collected by cervical spatula device or "thin preps" are transported in Cytyc proprietary preservative solution (buffered methanol) in 2ml polypropylene tubes. The samples are stable at room temperature for 3 weeks.
  • Washing of samples for use in the DNA amplification assay is performed as follows. An amount of 500 ⁇ l of wash buffer is added to a 500 ⁇ l aliquot of ThinPrep ® Cytyk sample and the resultant mixture is mixed by vortexing. The sample is then incubated at room temperature for 15 minutes.
  • the samples are centrifuged at approximately 12,500g for 5 minutes to form a pellet.
  • the sample preparation tubes are removed and the supernatant is carefully aspirated using a fine-tipped disposable pipette.
  • the pellet is then washed again with 500 ⁇ l of wash buffer and mixed well by vortexing.
  • the resulting mixture is centrifuged at approximately 12,500g for 5 minutes to form a pellet and then incubated at room temperature for 15 minutes.
  • Example 3 Sample preparation of C. trachomatis for qualitative PCR
  • Reagents are warmed to room temperature at least 30 minutes prior to sample preparation.
  • the positive C. trachomatis control is prepared from non- infectious C. trachomatis DNA in a Tris-HCl EDTA solution containing non-specific carrier DNA, less than 0.5% detergent and 0.05% sodium azide as a preservative, the negative C. trachomatis control can be an convenient DNA source other than C. trachomatis DNA in an equivalent mixture.
  • the PCR assay mixture comprises a Tris-HCl solution containing lOOmM EDTA, lOOmM KC1, 25% glycerol, 0.05% sodium azide (as a preservative), less than 0.016% dUTP, 0.01%
  • AmpliTaq® (Taq polymerase), less than 0.005% dATP, dCTP, and dGTP, less than 0.001% AmpErase, and 0.0004% biotinylated primers.
  • the prepared samples and controls can be stored at 2°C to 8°C for up to 7 days until ready for use (or is stable for 16 hours at room temperature).
  • a DNA amplification assay was carried out on samples of cells collected in an identical fashion to those analysed in the previous Examples.
  • the samples of cells were stored in buffered methanol medium (Cytyc) and were subject to DNA amplification assay without washing or pre-dilution and following the recommended protocol. The results are shown in Table 2.

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Abstract

A method is provided for the diagnosis for the presence of Chlamydia trachomatis in an individual by means of a DNA amplification assay, in which the sample cells are collected in a transport or collection medium for preservation and in which such cells can be processed for analysis using a DNA amplification assay directly.

Description

METHOD OF DIAGNOSIS FOR C. TRACHOMATIS INFECTION USING DNA AMPLIFICATION ASSAY ON LIQUID CYTOLOGY SAMPLES
The present invention relates to methods of diagnosis for C. trachomatis infection in a patient using DNA amplification assays to identify the presence of the infectious agent in a liquid cytology sample.
The early and accurate diagnosis of sexually transmitted diseases and cervical cancer are of major importance in the maintenance of good health for women, particularly with regard to female reproductive health. Diagnosis in women is important to be able to identify and treat infections to prevent further complications. Diagnosis in men is important principally in order to prevent further spread of the disease in question. Since the introduction of the cervico-vaginal Papanicolaou (Pap) smear test over fifty years ago there has been a dramatic reduction in the death rate for women from cervical cancer. Further recent improvements in the diagnosis of cervical cancer have concentrated on developments in the diagnostic method itself. The most significant recent development has been the ThinPrep® Pap Test™ of Cytyc Corporation (www . thinprep . com) which overcomes the problems associated with traditional smear tests by using a novel sample collection protocol. Instead of smearing collected cervical cells directly on a slide, the sampling device is rinsed into a collection vial containing a transport medium of buffered methanol,
PreservCyt®. The sample vial is then sent to a laboratory for analysis where slides are prepared from the liquid based sample. The improved method provides for the preparation of slides for analysis by automated means. The analysis of slides prepared by this method for the presence of atypical cells, cervical cancer or its precursor lesions is now the approved means for diagnosis in the USA by the Food & Drug Administration (FDA).
The ThinPrep® Pap Test™ has also now been licensed for use in the diagnosis of human papillomavirus (HPV) by Digene Corporation (www . di gene . com) . The HPV test uses the ThinPrep® sample vial produced by Cytyc Corporation containing PreservCyt® transport medium for the ThinPrep® Pap Test™ and analysis of the presence of HPV is carried out using the Digene Hybrid Capture HPV DNA Assay. This development was also important as nearly all cases of cervical cancer are positive for the presence of HPV. There are, though, other important diseases which if not diagnosed and treated early enough can lead to infertility problems for women. There is a need therefore to provide further improvements in sample collection and analysis to permit the easy, rapid and accurate diagnosis of conditions such as Chlamydia trachomatis and/or Neisseria gonorrhoeae. The use of DNA amplification assays such as the polymerase chain reaction
(PCR) in the diagnosis of HPV infection by Digene has suggested that other serious sexually transmitted diseases could also be diagnosed in the same way.
However, attempts to perform such analyses for Chlamydia trachomatis have failed as the DNA amplification assay does not provide a reliable or an accurate diagnostic result using the current protocols based on sample collection using a liquid based sample preparation approach.. For example Kellogg et al (J. Clin. Micobiol. 33(10) 2765-2767 (1995)), reported that PCR detection of C. trachomatis using PCR was significantly better with "dry" specimens than with specimens transported in a liquid sample collection medium.
Given the current popularity of the "wet" sample collection procedure for the diagnosis of cervical cancer and HPV using a single sample collected in a sample collection medium, there is therefore a need to provide a diagnostic assay for C. trachomatis using DNA amplification which overcomes the problems associated with the previously known methods.
It has now been surprisingly discovered that such methods can be made to work provided that the sample for analysis is prepared according to a method of the present invention which comprises effective washing of the cellular material to remove transport or storage medium in which the sample of cervical cells was collected and which further comprises pre-dilution (or titration) of cellular material in the sample prior to PCR hybridisation and detection reaction.
According to the present invention, there is provided a method for the diagnosis for the presence of Chlamydia trachomatis in an individual by means of a DNA amplification assay, comprising washing and pre-dilution of a sample of cells with a wash buffer that comprises a neutral buffer solution and a detergent, and optionally a halide salt of an alkali metal or an alkaline earth metal, in which the sample of cells is present in a sample collection medium after collection from the individual, wherein the washing and pre- dilution is carried out prior to lysis of the cells for the DNA amplification assay.
The effectiveness of the methods of the present invention was unexpected since attempts to diagnose C. trachomatis infections using PCR-based methods using sample collection medium, such as buffered methanol from Cytyc Corporation, had failed. An equivalent PCR-based diagnostic method had been seen to work previously in the identification of the presence of HPV in cervical cell samples prepared using Cytyc PreservCyt® liquid sample collection medium when the standard protocol had been followed. There had been an expectation that such methods would be applicable elsewhere. However, attempts up to the present date to replicate such assay procedures in the diagnosis of C. trachomatis were unsuccessful. The methods of the present invention now provide the solution to overcome this problem.
The presence of DNA from C. trachomatis in a sample as indicated by a valid, positive result from the DNA amplification assay may indicate the presence of a systemic infection by the organism in the individual being tested, or it may indicate recent introduction of the organism to the individual through close personal contact with another infected individual..
The DNA amplification assay may be the Polymerase Chain Reaction (PCR). One suitable method based on the PCR approach is the automated COBAS AMPLICOR™ system of Roche Diagnostics (F. Hoffman-La Roche Ltd. - www.roche.com). The DNA of C. trachomatis to be amplified is extracted from chlamydial reticulate bodies obtained from cells sampled from an individual patient. Other PCR-based amplification methods can be selected as appropriate by a person skilled in the art.
Suitable methodologies for carrying out DNA amplification assays can be found in "PCR Primer: A Laboratory Manual", C. W. Diffenbach & G. S. Dveksler, eds., Cold Spring Harbor Laboratory Press (1995), "The Polymerase Chain Reaction", K. B. Mullis et al, Birkhauser (1994), "PCR Strategies", M. A. Innis et al, eds., Academic Press (1995), and
"PCR: A Practical Approach", M. J. McPherson et al, eds., IRL Press (1991). Primers and probes for DNA amplification of DNA samples from C trachomatis can be prepared according to Pudijiatmoko et al in Int. J. Syst. Syst. Bacteriol. 41{2), 425-431 (1997). For example, the 16S rRNA of C. trachomatis can be used in order to design primers for use in DNA amplification assays (Song et al Combinatorial Chemistry & High Throughput Screening, 3(4), (2000)). The genome of C. trachomatis is now available at www.stdgen.lanl.gov or Los Alamos National Laboratory Bioscience Division.
The cells present in the sample from the individual to be assayed may be from any site (or potential site) of infection in an individuals body. Typically, in a method of the present invention practised on a female, the cells will be of cervico-vaginal origin and will be epithelial cells.
The washing of the sample may be undertaken by any suitable means to remove the sample collection medium. Typically, though, the washing of the sample may comprise addition of wash buffer to the sample present in the sample collection medium, followed by centrifugation to form a pellet. An aliquot of cells in sample collection medium is washed with an equivalent aliquot of wash buffer. Generally, the aliquots will be in the ratio of 1 : 1 and this can be varied as required up to an addition of wash buffer in excess in order to complete full washing of the cells in the sample to remove sample collection medium.
The wash buffer may comprise any suitable neutral buffer and detergent, optionally including saline, that can preserve the integrity of the cells in the sample whilst permitting washing out of the sample collection medium. A buffer can be defined as a substance which when added to a solution resists a change in hydrogen ion concentration on addition of acid or alkali (Grant & Hackh's Chemical Dictionary, McGraw-Hill Book Company, 5th edition (1987)).
A neutral pH is typically about pH 7.0, however the effective pH range of the buffer solutions of the present invention may be in the range of from pH6.9 to pH8.2, suitably pH7.4 to pH7.8, where a convenient working pH may be pH7.6.
Suitable buffers include but are not limited to buffer solutions prepared from tris(hydroxymethyl)aminomethane (or tris base or Trizma® base), or its hydrochloride equivalent (Tris HC1 or Trizma® HC1). Tris buffered saline tablets may also be used to prepare alternative versions of the wash buffer that contain saline in the form of sodium chloride.
Other suitable buffers that provide neutral buffer solutions are Aces (N-
[Carbamoylmethyl]-2-aminoethanesulfonic acid or N-[2-Acetamidol-2-aminoethane sulfonic acid), Pipes (Piperazine-N, N'-bis[2-ethanesulfonic acid or 1 ,4-Piperazinediethane sulfonic acid), Mopso (3-[N-Mo holino]-2-hydroxypropane sulfonic acid), Bis-Tris Propane (l,3-bis[tris(Hydroxymethyl)methylamino]propane), Bes (N, N-bis[2- Hydroxyethyl]-2-aminoethanesulfonic acid), Mops (3-[N-Morpholino]propane sulfonic acid), Tes (N-tris{Hydroxymethyl]methyl-2-aminoethanesulfonic acid or 2-([2-Hydroxy- l,l-bis(hydroxymethyl)-ethyl]amino)sulfonic acid), Hepes (N-[2-Hydroxyethyl] piperazine-N'-[2-ethane sulfonic acid]), Dipso (3-[N, N-bis(2-Hydroxyethyl)amino]-2- hydroxy-propane sulfonic acid), Mobs (4-[N-Morpholino]butane sulfonic acid), Tapso (3- [N-tris(hydroxymethyl)methylamino]-2-hydroxy-propane sulfonic acid), Heppso (N-[2-
Hydroxyethyl]piperazine-N'-[2-hydroxy-propane sulfonic acid]), Popso (Piperazine-N, N'- bis[2-hydroxypropane sulfonic acid]), TEA or triethanolamine (with or without magnesium EDTA), Epps (N-[2-Hydroxyethyl]piperazine-N'-[3-propane sulfonic acid] or Hepps), Tricine (N-tris[Hydroxymethyl]methylglycine or N-[2-Hydroxy-l,l-bis(hydroxymethyl) ethyl] glycine), Glycyl glycine or Gly-Gly, Bicine (N, N-bis[2-Hydroxyethyl]glycine),
Hepbs (N-[2-Hydroxyethyl]piperazine-N'-[4-butanesulfonic acid] which is a homolog of Hepes and Epps with a higher pKa), or Taps (N-tris[Hydroxymethyl]methyl-3- aminopropane sulfonic acid or [2-Hydroxy-l,l-bis(hydroxymethyl)ethyl]amino)-l-propane sulfonic acid).
The molarity of the buffer may be in the range of from lOmmol/L to 500mmol/L, preferably from 25mmol/L to 150mmol/L, suitably from 50mmol/L to lOOmmol/L.
The detergent substance added to the buffer solution may be any convenient detergent that can preserve the integrity of the cells in the sample whilst permitting washing out of the sample collection medium. Suitably, the detergent is a non-ionic detergent. The detergent may be present in the range of from 0.05% to 0.25%, preferably 0.075% to 0.2%, or at 0.1%. Suitable detergents, include but are not limited to t-octylphenoxypolyethoxy ethanol (Triton®) or polyoxyethylenesorbitan monolaurate (Tween®). Other non-ionic detergents are BIGCHAP, decanoyl-N-methylglucamide, n-Decyl α-D-glucopyranoside, n-Decyl β- D-glucopyranoside, n-Decyl β-D-maltopyranoside, DeoxyBIGCHAP, n-Dodecyl β-D- glucopyranoside, n-Dodecyl α-D-maltoside, n-Dodecyl β-D-maltoside, Heptanoyl-N- methyl glucamide, n-Heptyl β-D-glucopyranoside, N-Heptyl β-D-thioglucopyranoside, n- Hexyl β-D-glucopyranoside, Igepal CA-630, 1-Monooleolyl-rac-glycerol, Nonanoyl-N- methyl glucamide, n-Nonyl α-D-glucopyranoside, n-Nonyl β-D-glucopyranoside, Octanoyl-N-methylglucamide, n-Octyl α-D-glucopyranoside, n-Octyl β-D- glucopyranoside, Octyl β-D-Thiogalactopyranoside, Octyl β-D-Thioglucopyranoside, Polyoxyethylene esters, polyoxyethylene ethers, polyoxyethylenesorbitan esters, sorbitan esters, Tergitol, n-Tetradecyl β-D-maltoside, Tyloxapol, or n-Undecyl β-D- Glucopyranoside.
• • • (R) t-octylphenoxypolyethoxy ethanol can be provided in different forms such as Tπton X- 100, Triton® X-100 Reduced, Triton® X-114, Triton® X-405, Triton® X-405 Reduced, Triton® CG-110, Triton® XL-80N, Triton® WR-1339 or Tyloxapol. Other non-ionic Triton® detergents are N-42, N057, N-60, X-15, X-35, X-45, X-102, X-155, X-165, X-207, X-305, X-705-70, B-1956. In many situations, Triton® X-100 may be preferable.
Polyoxyethylenesorbitan monolaurate can be provided in different forms such as Span 20, Tween® 20, Tween® 20R, Tween® 21, Tween® 80, Tween® 80R, Tween® 80K, Tween® 81, Tween® 40, Tween® 60, Tween® 61, Tween® 85, or Tween® 65. Suitably, the polyoxyethylenesorbitan monolaurate can be Tween® 20.
The halide salt of an alkali metal or an alkaline earth metal may be a fluoride, a chloride, a bromide or an iodide salt. The alkali metal ion may be lithium sodium, or potassium. The alkaline earth metal ion may be magnesium, or calcium. In certain preferred embodiments of the invention, the halide salt is sodium chloride. The halide salt may be selected from the list of sodium fluoride, sodium chloride, potassium chlorides, magnesium chloride, or calcium chloride. Sodium chloride may optionally be included in the composition of the buffer in a range of from 50mmol/L to 500mmol/L, suitably lOOmmol/L to 400mmol/L, preferably from 150mmol/L to 300mmol/L.
The pre-dilution or titration of the sample may be by any convenient means to dilute out cellular material. Typically, though, the pre-dilution or titration step may comprise addition of wash buffer to the sample after washing described above.
The sample of cells taken from the individual patient are collected by any generally convenient means dependent upon the location of the cells to be assayed. For example, a gynaecological sample may be collected using a broom-type or a cytobrush spatula cervical sampling device. The cells obtained are then rinsed into a sample collection vial containing sample collection medium. In certain preferred embodiments of the invention, the medium is buffered methanol, for example PreservCyt® medium. Where the medium is buffered methanol, it may be suitably buffered to a neutral pH as described in detail above in realtion to the wash buffer.
Lysis of the cells of the individual and reticulate bodies of the C. trachomatis in the sample is necessary to retrieve DNA of the infectious agent. Lysis can be achieved by any suitable means, but typically may involve application of a lysis medium, for example a detergent.
In one preferred embodiment of the present invention, there is provided a method for the diagnosis for the presence of Chlamydia trachomatis in an individual by means of a DNA amplification assay using the COBAS AMPLICOR™ system of Roche Diagnostics (F. Hoffman-La Roche Ltd. - www.roche.com). The method comprises washing a sample of cells with a wash buffer comprising a neutral buffer solution of tris(hydroxymethyl) aminomethane and a detergent selected from t-octylphenoxypolyethoxy ethanol or polyoxyethylenesorbitan monolaurate, with sodium chloride present in the medium. The sample of cells to be washed is present in a sample collection medium, typically buffered methanol, after collection from the individual, and wherein the washing is carried out prior to lysis of the cells for the DNA amplification assay. In a second aspect of the invention there is provided a method for the diagnosis for the presence of Chlamydia trachomatis in an individual by means of a DNA amplification assay which may be considered to comprise the steps of
(a) collecting sample of cervical cells; (b) introducing said sample of cells into a collection vial containing sample collection medium;
(c) introducing wash medium to sample;
(d) centrifuging to form pellet;
(e) removing supernatant from pellet; (f) introducing wash medium to pellet and resuspending;
(g) centrifuging to form pellet;
(h) adding cell lysis medium to pellet;
(i) resuspending pellet;
(j) centrifuging to form pellet; (k) removing supernatant;
(1) dilution of supernatant with wash buffer; and
(m) analysis by amplification assay for C. trachomatis DNA.
Previous attempts to carry out DNA amplification assays for C. trachomatis DNA have failed because the wash and dilution steps (c) to (1) were not performed. Without wishing to be bound by theory, it is presumed that the remaining presence of sample collection medium and high levels of cellular material interferes with the DNA amplification assay such that results cannot be obtained.
In operation, a method of the present invention may comprise therefore a means for preparing a sample of cells taken from an individual for the purposes of diagnosing C. trachomatis infection, in which the sample of cells is preserved in a sample collection medium after collection from the individual, suitably the buffered methanol solution known as PreservCyt® produced by Cytyc Corporation for the ThinPrep® Pap Test™. Preservation of the sample in sample collection medium may for the purposes of preservation prior to analysis, including transportation from the site of collection to the site of analysis. The sample may also be used to provide cells for the diagnosis of other medical conditions such as, for example, cervical cancer and/or human papillomavirus (HPV) infection.
Once the sample of cells is to be assayed for the presence of C. trachomatis DNA, the sample needs to be prepared according to a method of the present invention prior to the use of the DNA amplification assay. In a method of the present invention, an aliquot of wash medium is introduced to the sample; or to an aliquot from the original sample in an appropriate vessel or tube. The mixture is then centrifuged in the vessel or tube to form a pellet. The supernatant from pellet, such as for example by aspiration using a pipette. A further aliquot of wash medium is then introduced to the pellet which is resuspended in this medium by, for example, vortexing. The resulting mixture is then re-centrifuged to form a pellet which is suitable for processing for the DNA amplification assay. This is achieved by adding cell lysis medium to pellet and resuspending the pellet in this medium. Finally, centrifugation of this mixture yields a supernatant that can be removed and pre-diluted (titrated) for analysis by amplification assay for C. trachomatis DNA
According to a third aspect of the invention there is provided a kit of parts for the diagnosis of Chlamydia trachomatis infection in a sample from an individual by means of a DNA amplification assay, the kit comprising a wash buffer that comprises a neutral buffer solution and a detergent, and optionally a halide salt of an alkali metal or an alkaline earth metal.
Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.
Example 1: Preparation of Wash buffer
The wash buffer is prepared from Tris-HCl or Tris base (Sigma) to molarity lOOmmol/L at pH7.6. The solution is completed by the addition of t-octylphenoxy polyethoxyethanol (Sigma) to 0.1% and sodium chloride to 150mmol/L.
Example 2: Washing of cells from collected samples Samples collected by cervical spatula device or "thin preps" are transported in Cytyc proprietary preservative solution (buffered methanol) in 2ml polypropylene tubes. The samples are stable at room temperature for 3 weeks.
Washing of samples for use in the DNA amplification assay is performed as follows. An amount of 500μl of wash buffer is added to a 500μl aliquot of ThinPrep® Cytyk sample and the resultant mixture is mixed by vortexing. The sample is then incubated at room temperature for 15 minutes.
After incubation, the samples are centrifuged at approximately 12,500g for 5 minutes to form a pellet. The sample preparation tubes are removed and the supernatant is carefully aspirated using a fine-tipped disposable pipette. The pellet is then washed again with 500μl of wash buffer and mixed well by vortexing. The resulting mixture is centrifuged at approximately 12,500g for 5 minutes to form a pellet and then incubated at room temperature for 15 minutes.
Example 3: Sample preparation of C. trachomatis for qualitative PCR
Reagents are warmed to room temperature at least 30 minutes prior to sample preparation.
At the time of sample preparation, appropriate positive and negative C. trachomatis control samples are also prepared. The positive C. trachomatis control is prepared from non- infectious C. trachomatis DNA in a Tris-HCl EDTA solution containing non-specific carrier DNA, less than 0.5% detergent and 0.05% sodium azide as a preservative, the negative C. trachomatis control can be an convenient DNA source other than C. trachomatis DNA in an equivalent mixture.
Samples of cells are washed and prepared as described in Example 2 above. Following sample preparation, samples and controls are then mixed with an aliquot of the PCR assay mixture. For each 50μl of sample 50μl of assay mixture is mixed thoroughly. The PCR assay mixture comprises a Tris-HCl solution containing lOOmM EDTA, lOOmM KC1, 25% glycerol, 0.05% sodium azide (as a preservative), less than 0.016% dUTP, 0.01%
AmpliTaq® (Taq polymerase), less than 0.005% dATP, dCTP, and dGTP, less than 0.001% AmpErase, and 0.0004% biotinylated primers. The prepared samples and controls can be stored at 2°C to 8°C for up to 7 days until ready for use (or is stable for 16 hours at room temperature).
Following addition of PCR assay mixture, the sample is vortexed thoroughly prior centrifugation at approximately 12,500g for 10 minutes. Finally, addition to the sample of 250μl lysis buffer composed of a Tris-HCl solution containing approximately 1% solubiliser and 0.09% sodium azide as a preservative. The mixture is vortexed well and incubated for 10 minutes at room temperature and is then ready for PCR assay using COBAS AMPLICOR™ system of Roche Diagnostics (F. Hoffman-La Roche Ltd. - www.roche.com). Results of assays using this method of sample preparation are shown in Table 1.
Table 1
Figure imgf000012_0001
These results compare very favourably to the results of the prior art methods, shown in the following comparative Example.
Comparative Example 1: Results of prior art methods
A DNA amplification assay was carried out on samples of cells collected in an identical fashion to those analysed in the previous Examples. The samples of cells were stored in buffered methanol medium (Cytyc) and were subject to DNA amplification assay without washing or pre-dilution and following the recommended protocol. The results are shown in Table 2.
Table 2
Figure imgf000013_0001

Claims

1. A method for the diagnosis for the presence of Chlamydia trachomatis in an individual by means of a DNA amplification assay, comprising washing and pre-diluting a sample of cells with a wash buffer comprising a neutral buffer solution and a detergent, and optionally a halide salt of an alkali metal or an alkaline earth metal, in which the sample of cells is present in a sample collection medium after collection from the individual, wherein the washing and pre-dilution is carried out prior to lysis of the cells for the DNA amplification assay.
2. A method as claimed in claim 1, in which the DNA amplification assay is the Polymerase Chain Reaction (PCR).
3. A method as claimed in claim 1 or claim 2, in which the cells present in the sample from the individual to be assayed are epithelial cells.
4. A method as claimed in any of claims 1 to 3, in which the pH range of the neutral buffer solution is in the range of from pH6.9 to pH8.2
5. A method as claimed in any of claims 1 to 4, in which the detergent is a non-ionic detergent.
6. A method as claimed in any preceding claim, in which the wash buffer is prepared from tris(hydroxymethyl)aminomethane or the hydrochloride equivalent
7. A method as claimed in any preceding claim, in which the molarity of the buffer is in the range of from lOmmol/L to 500mmol/L, preferably from 25mmol/L to 150mmol/L, suitably from 50mmol/L to lOOmmol/L.
8. A method as claimed in any preceding claim, in which the detergent substance added to the buffer solution is present in the range of from 0.05% to 0.25%, suitably 0.075% to 0.2%, or at 0.1%.
9. A method as claimed in any one of claims 5 to 8, in which the detergent is selected from the group consisting of t-octylphenoxypolyethoxy ethanol or polyoxyethylenesorbitan monolaurate.
10. A method as claimed in any preceding claim, in which the halide salt of an alkali metal or an alkaline earth metal comprises sodium fluoride, sodium chloride, potassium chlorides, magnesium chloride, or calcium chloride.
11. A method as claimed in claim 10, in which sodium chloride is present in the buffer at a concentration in a range of from 50mmol/L to 500mmol/L, suitably lOOmmol/L to
400mmol/L, preferably from 150mmol/L to 300mmol/L.
12. A method as claimed in any preceding claim in which the sample collection medium is buffered methanol.
13. A method for the diagnosis for the presence of Chlamydia trachomatis in an individual by means of a DNA amplification assay, comprising the steps of
(a) collecting sample of cervical cells;
(b) introducing said sample of cells into a collection vial containing sample collection medium;
(c) introducing wash medium to sample;
(d) centrifuging to form pellet;
(e) removing supernatant from pellet;
(f) introducing wash medium to pellet and resuspending; (g) centrifuging to form pellet;
(h) adding cell lysis medium to pellet; (i) resuspending pellet; (j) centrifuging to form pellet; (k) removing supernatant; (1) dilution of supernatant with wash buffer; and
(m) analysis by amplification assay for C. trachomatis DNA.
14. A kit of parts for the diagnosis of Chlamydia trachomatis infection in a sample from an individual by means of a DNA amplification assay, the kit comprising a wash buffer that comprises a neutral buffer solution and a detergent, and optionally a halide salt of an alkali metal or an alkaline earth metal.
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WO2007149350A2 (en) * 2006-06-19 2007-12-27 Becton, Dickinson And Company Methods and compositions for obtaining amplifiable nucleic acids from tissues, cells, or viruses exposed to transport media
WO2007149350A3 (en) * 2006-06-19 2008-03-20 Becton Dickinson Co Methods and compositions for obtaining amplifiable nucleic acids from tissues, cells, or viruses exposed to transport media
JP2009540827A (en) * 2006-06-19 2009-11-26 ベクトン・ディキンソン・アンド・カンパニー Methods and compositions for obtaining amplifiable nucleic acids from tissues, cells, or viruses exposed to transport media
AU2013205379B2 (en) * 2006-06-19 2016-03-10 Becton, Dickinson And Company Methods and compositions for obtaining amplifiable nucleic acids from tissues, cells, or viruses exposed to transport media
US10190152B2 (en) 2009-09-03 2019-01-29 Becton, Dickinson And Company Methods and compositions for direct chemical lysis
US10323267B2 (en) 2009-09-03 2019-06-18 Becton Dickinson And Company Methods and compositions for direct chemical lysis
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CN105925692A (en) * 2016-05-16 2016-09-07 安陆市疾病预防控制中心 Normal-temperature stable PCR premixed solution
CN105925692B (en) * 2016-05-16 2019-09-20 安陆市疾病预防控制中心 A kind of PCR premixed liquid of ambient stable

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