Molecular and Biochemical Parasitology, 43 (1990) 69-76 Elsevier
69
MOLBIO 01411
Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin A n n E. Wakefield 1, Fiona J. Pixley 1, Suneale Banerji j , Kathryn Sinclair I , Robert F. Miller 2, E. Richard M o x o n 1 and Julian M. Hopkin 3 Ilnstitute of Molecular Medicine, John Radcliffe Hospital, Oxford, 2Department of Medicine, UCMSM, Middlesex Hospital, London, and 3Osier Chest Unit, Churchill Hospital, Oxford, U.K. (Received 12 April 1990; accepted 30 May 1990)
Pneumocystis carinii specific DNA sequences have been cloned from the experimental rat model. The sequence of the gene coding for the large subunit of mitochondrial ribosomal RNA has been used to construct P. carinii specific oligonucleotide primers for the polymerase chain reaction. These oligonucleotides produced amplification of specific sequences from both P. carinii infected rat and human lung samplings, but none from a range of other organisms including potential pulmonary pathogens. Comparison of the sequence of amplified products from the infected rats and humans demonstrated limited but consistent differences between P. carinii from these two hosts and allowed for the construction of a human specific internal oligonucleotide. The application of the specific oligonucleotides for DNA amplification and subsequent Southern hybridisation affords extremely sensitive and specific detection of P. carinii in human samples, which ma3/be applicable to both epidemiological research and clinical studies. Key words: Pneumocystis carinii; DNA amplification
Introduction
Pneumocystis carinii is established as the prime cause of opportunistic pneumonia in patients with AIDS and those immunosuppressed on oncology and transplant units. Uncertainties about the parasite's taxonomy and the epidemiology of infection continue and are relevant to strategies for improving the prevention and treatment of this disease. The fastidious nature of P. carinii still demands the use of microscopy to identify the parasite in different samplings, after histochemical staining [1], with its inherent non-specificity or, more recently, immunofluorescence [2] of uncertain sensitivity [3]. There are sound theoretical grounds for believing that DNA amplification using the polymerase chain reaction (PCR) [4] might proCorrespondence address: Ann E. Wakefield, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, U.K. Abbreviations: PCR, polymerase chain reaction; bp, base pair.
vide both a highly specific and highly sensitive means of identifying the parasite. Materials and Methods
Cloning and sequencing of part of the gene coding for the large subunit of the mitochondrial ribosomal RNA from P. carinii. P. carinff pneumonia was induced in the rat model and DNA extracted and cloned from a parasite enriched fraction as previously described [5]. P. carinii specific sequences were confirmed by characteristic in situ " hybridisation patterns and recombinant plasmid pAZ102 was selected as a candidate mitochondrial sequence because of strong signals derived in dot blot hybridisation studies on infected samples. The recombinant plasmid pAZ102 (insert 570 bp) was sequenced using Sanger's chain termination method and the Sequenase kit (United States Biochemical Corporation, Cleveland, OH, U.S.A.), 35S (Amersham, U.K.), Sequagel (National Diagnostics, Manville, U.S.A.). The DNA sequence was compared with those available in
0166-6851/90/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
70
Lanes- 1
2
3
4
5
6
7
8
9
(b)
(c)
• • e~
B~
71 Lan6s-
1
2
4
6
8
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12
14
16
18 19 20
(b)
t
'----"--
•
Fig. 2. Results of DNA amplification using P. carinii specific oligonucleotide primers applied to DNA templates from P. carinii infected human lung and other organisms including potential pulmonary pathogens. Products of DNA amplification, using DNA templates prepared from: lane 2, rat; lane 4, human; lane 6, Candida albicans; lane 8, Candida (non-albicans); lane 10, Cryptococcus neoformans; lane 12, Mycobacterium tuberculosis; lane 14, Saccharomyces cerevisiae; lane 16, Aspergillus nidulans; lane 18, human-derived P. carinii. Lane 19, no template control; lanes 1 and 20: 1-kb molecular weight markers. (A) Ethidium bromide stained gel. (B) Autoradiograph of Southern blot probed with pAZI02-L2. Asterisks mark arrows pointing to bands of amplified DNA specific to P. carinii.
Fig. 1. Amplified DNA sequences demonstrated by ethidium bromide staining in pulmonary samplings from both rat and human hosts. Comparative oligoblotting with rat and human P. carinii specific internal oligonucleotide. Products of DNA amplification using DNA templates prepared from: lanes 2, 3, 4, rat-derived P. carinii; lanes 5, 6, 7, human-derived P. carinii. Lane 8, no template control; lanes 1 and 9, 1-kb molecular weight markers. (A) Ethidium bromide stained gel. (B) Autoradiograph of Southern blot probed with pAZI02-LI; (C) Autoradiograph of Southern blot probed with pAZ102-L2. Asterisks mark arrows pointing to bands of amplified DNA specific to P. carinii.
72 several databases including EMBL and GenBank. From the sequence data on pAZ102 and comparative analysis of the databases, the fragment was identified as a portion of the gene coding for the large subunit of the mitochondrial, ribosomal RNA of P. carinii and this showed significant homology with fungal sequences (manuscript in preparation).
Oligonucleotide primers. Two sequences of moderate conservation that were specific to P. carinii were selected for construction of oligonucleotide primers for the polymerase chain reaction: pAZ 102-E: -5'-GATGGCTGTTTCCAAGCCCA3'; pAZI02-H:-5'-GTGTACGTTGCAAAGTACTC-3'. An oligonucleotide for confirmatory Southern hybridisation on amplification products was chosen, pAZ102-L1. Subsequently a new internal oligonucleotide specific to human P. carinii sequences was constructed, pAZ102-L2 (Fig. 5). Template DNA. (i) Samplings for DNA amplification using our oligonucleotide primers comprised (a) pulmonary lavage samplings from 3 humans and 3 rats with P. carinii pneumonia documented by methenamine silver staining and microscopy, and (b) isolates from a series of organisms including some potential pulmonary pathogens: Candida (an albicans and a non-albicans strain), Cryptococcus neoformans, Mycobacterium tuberculosis, Saccharomyces cerevisiae and Aspergillus nidulans (Fig. 2). (ii) Template DNA was prepared from each sample by proteinase K digestion in the presence of SDS and EDTA followed by phenol/chloroform/ether extraction, and ethanol precipitation. DNA amplification. Using primers pAZ102-E and pAZ102-H the template samples, together with control samples without template, underwent 40 cycles of amplification performed with denaturation at 94°C for 90 s, annealing at 50°C for 90 s and extension at 72°C for 2 min (Techne, U.K.). The DNA amplification reaction mixture (50 11,1) contained 50 mM KC1/10 mM Tris, pH 8/0.01% (w/v) gelatin/3 mM MgC12/400 #M dNTPs (Boehringer Mannheim, U.K.)/0.4-1.0 #M
oligonucleotide/3 units of Amplitaq (Perkin Elmer Cetus, U.K.). To avoid the possibility of false negative results in the human clinical samples, i.e., failure to detect the specific amplification product for technical reasons, we carried out a parallel polymerase chain reaction on each sample using primers derived from the human anti-thrombin gene, exon 2 (S.L. Thein, personal communication). These primers, (ATI:-5'GTTGCAGCCTAGCTTAACTTGGCA- 3'; AT4:5'-GGT/'GAGGAATCATTGGACTTG-3') allowed amplification to take place using human genomic DNA as template. In each of the clinical samples the 500-bp specific product was detected, demonstrating efficient amplification. The potential problem of contamination in PCR was monitored by systematic use of the following techniques: (i) including several negative control samples with no added template DNA; (ii) by the use of UV irradiation of the PCR reaction mixes prior to the addition of the template DNA [6]; (iii) the use of separate disposable microcapillaries for the addition of each template (Laser Laboratory Systems Ltd., U.K.). The control samples remained negative in all experiments. Amplified products (10 #1) were electrophoresed in 1.5% agarose gels and visualised after ethidium bromide staining by ultraviolet light. The gel was Southern blotted on to Hybond N (Amersham, U.K.) and hybridised with 32p end-labelled internal primer at 46°C (pAZI02-L1) or 40°C (pAZ102-L2) for 3 h [7]. Filters were subsequently washed at high stringency at 54°C (pAZ102-L1) or 48°C (pAZ102-L2) and filters exposed to radiographic film at - 8 0 ° C with intensifying screens. The expected amplification product was 355 bp long in the rat derived parasite, that from the human derived parasite being 9 bp shorter.
Sequencing of ppvducts of DNA amplification. The PCR product was gel-purified and recovered from the agarose gel using Geneclean (Bio 101, Inc). The purified DNA was heat-denatured and sequenced as described above using primers pAZ102-H or pAZ102-E at 20 pmol//~l.
73
Lanes- 1 2
4
6
8
10
12
17
19
(b)
O
Fig. 3. Sensitivity of detection of amplification products using ethidium bromide staining and oligoblotting. Products of amplification using serially diluted DNA template prepared from 2 ml bronchoalveolar lavage from a patient with confirmed P. carinii infection. Lane 2, undiluted; lane 4, diluted x 10; lane 6, diluted x 102; lane 8, diluted × 103; lane 10, diluted x 104; lane 12, diluted x 105. Lane 17, no template control; lanes 1 and 19, l-kb molecular weight markers. (A) Ethidium bromide stained gel. (B) Autoradiograph of Southern blot probed with pAZI02-L2. Asterisks mark arrows pointing to bands of amplified DNA specific to P. carinii. Results
The oligonucleotide primers derived from rat P. carinii produced efficient amplification of specific
sequence from both rat and h u m a n hosts, shown by ethidium bromide staining (Fig. 1) but none
from the range of other organisms including s o m e potential p u l m o n a r y pathogens (Fig. 2). The internal oligonucleotide, p A Z 1 0 2 - L 1 , derived from the rat P. carinii, produced strong hybridisation signals on Southern hybridisation with amplified products from the infected rat lungs, but
74
Lanes l
2
4
~iiF
~ii!i!i
6
8
10
12
14
16
18
20
22
24 25 2627 28
30
(a)
1,018~,
344'~' 298,~
(b)
Fig. 4. DNA amplification from a series of human samplings and oligoblotting with human derived P. carinii internal oligonucleotide pAZ102-L2. Products of DNA amplification using DNA template prepared from 2 ml bronchoalveolar lavage sampling: lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, patients with confirmed P. carinii infection; lanes 24, 25, 26, 27, 28, immunocompetent patients without P. carinii infection; lanes I, 22, 30, l-kb molecular weight markers. (A) Ethidium bromide stained gel. (B) Autoradiograph of Southern blot probed with pAZI02-L2. Asterisks mark arrows pointing to bands of amplified DNA specific to P. carinii.
weak signal, at high stringency, with the amplified product derived from human samples although these were visible on ethidium bromide staining (Fig. 1). Direct sequencing of the amplified products from each of the rat and human samples allowed comparison of their sequence and demon-
strated limited but consistent differences between the P. carinii DNA from these two hosts which included 5 base changes in the sequence of the internal oligonucleotide pAZ102-L1 (Fig. 5). An oligonucleotide specific to the human derived organisms was constructed, pAZ102-L2,
75
pAZI02-L2
5'
A A G G
A G A T
A
Fig. 5. Comparative sequence of oligonucleotides pAZI02-LI (rat P. carinii) and pAZI02-L2 (human P. carinii).
which showed strong hybridisation with the amplified product from human P. carinii and conversely showed weak hybridisation with the rat P. carinii amplified product (Fig. 1). It produced no hybridisation with the PCR products of the range of other organisms tested (Fig. 2). Studies using serial dilutions of human derived P. carinii template DNA indicated that the application of oligoblotting with pAZ102-L2 to amplified DNA products increased the sensitivity of detection by at least 100-fold over visualisation by ethidium bromide staining (Fig. 3). The P. carinii oligonucleotide primers successfully amplified specific PCR product from bronchoscopic alveolar lavage samplings from 10 HIVpositive individuals with pneumocystis pneumonia as documented by positive methenamine silver staining on the lavage samples (Fig. 4). Lavage samples from 5 immunocompetent subjects were studied as controls. These failed to show specific PCR product by ethidium bromide staining or oligoblotting.
Discussion We have characterised a portion of the gene coding for the large subunit of mitochondrial, ribosomal RNA from P. carinii and comparative analysis of this indicates significant homology with fungal sequences. This result accords with data we have on other mitochondrial genes (manuscript in preparation) and the observations of other groups on ribosomal RNA [8]. We have identified P. carinii specific sequences from which we have constructed oligonucleotide primers which allow efficient amplification of part of this ribosomal RNA gene from P. carinii infecting both rat and human hosts but not from a range of other organisms including some potential pulmonary pathogens. These results indicate the specificity of the amplified products to P. carinii, confirmed on Southern hybridisation with internal oligonucleotide pAZI02-LI derived from the rat
sequence and applied to the rat pulmonary samplings. Results from the human samplings suggested the likelihood of differences in sequenEe between the amplified products from the rat and human. This was confirmed by comparison of sequences which indicated limited, but consistent differences. This finding is highly relevant to any hopes of applying DNA amplification for specific sensitive detection of P. carinii in various samplings. We have shown that our oligonucleotide primers can be used to identify the presence of P. carinii in a number of human bronchoalveolar lavage samples. By using our second internal oligonucleotide, pAZ102-L2, which is specific to human P. carinii, the sensitivity of detection of amplified product is increased 100-fold. Thus amplified P. carinii specific DNA is detectable in lavage diluted × 105 from patients with documented P. carinii pneumonia. In contrast, we found no P. carinii DNA in lavage from immunocompetent subjects, thus questioning further the current dogma that opportunistic pneumonia results necessarily from reactivation of latent pulmonary infection by the parasite [9]. DNA amplification and subsequent oligoblotting offers a method that combines high specificity and sensitivity for the detection of the parasite. This should be immediately applicable to the studies now required to address the unresolved epidemiology of this infection and the potential exists for the technique to be developed, by suitable calibration and automation now available [ 10,11], for the accurate diagnostic work required for therapeutic studies.
Acknowledgements We thank Professor S.J.G. Semple for the HIV positive lavage samples; Dr. S.L. Thein and Dr. R. Olds for the human anti-thrombin primers; and Dr. D. Young for the Mycobacterium tuberculosis DNA. This work was supported by the Royal Society (AEW), the Medical Research Council (FJP) and the Wellcome Trust (SB and KS). The sequence of pAZ102 is subject to patent applications.
76
References 1 Savoia, D., Carmello, P. (1989) The microscopic identification of Pneumocystis carinii. J. Protozool. 36, 72S-74S. 2 Kovacs, J.A., Ng, V.L., Masur, H., Leoung, G., Hadley, W.K., Evans, G., Lane, H.C., Ognibene, F.P., Shelhamer, J., Parillo, J.E. and Gill, V.J. (1988) Diagnosis of Pneumocystis carinii pneumonia: improved detection in sputum with use of monoclonal antibodies. New Engl. J. Med. 318, 589-593. 3 Linder, E., Lundin, L. and Vorma, H. (1987) Detection of P. carinii in lung derived samples using monoclonal antibodies to an 82-kDa parasite component. J, lmmunol. Methods 98, 57--62. 4 Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985) Enzymatic amplification of/4-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 230, 1350-1354. 5 Wakefield, A.E., Hopkin, J.M., Bums, J., Hipkiss, J.B.,
6 7 8
9
10 11
Stewart, T.J. and Moxon, E.R. (1988) Cloning of DNA from Pneumocystis carinii. J. Infect. Dis. 158. 859-861. Sarkar, G. and Sommer, S.S. (1990) Shedding light on PCR contamination. Nature 343, 27. Miyada, C.G. and Wallace, R.B. (1987) Oligonucleotide hybridization techniques. Methods Enzymol. 154, 94-107. Edman, J.C., Kovacs, J.A., Masur, H., Santi, D.V., Elwood, H.J. and Sogin, M.L. (1988) Ribosomal RNA sequence shows Pneumocystis carinii to be a member of the Fungi. Nature 334, 519-522. Millard, P.R. and Heryet, A.R. (1988) Observations favouring Pneumocystis carinii pneumonia as a primary infection: a monoclonal antibody study on paraffin sections. J. Pathol. 154, 365-370. Wang, A.M., Doyle, M.V. and Mark, D.F. (1989) Quantitation of mRNA by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86, 9717-9721. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Quantitation of initial concentration of target sequences. In: Molecular Cloning (2nd ed.), pp 14.30-14.35, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
69
MOLBIO 01411
Amplification of mitochondrial ribosomal RNA sequences from Pneumocystis carinii DNA of rat and human origin A n n E. Wakefield 1, Fiona J. Pixley 1, Suneale Banerji j , Kathryn Sinclair I , Robert F. Miller 2, E. Richard M o x o n 1 and Julian M. Hopkin 3 Ilnstitute of Molecular Medicine, John Radcliffe Hospital, Oxford, 2Department of Medicine, UCMSM, Middlesex Hospital, London, and 3Osier Chest Unit, Churchill Hospital, Oxford, U.K. (Received 12 April 1990; accepted 30 May 1990)
Pneumocystis carinii specific DNA sequences have been cloned from the experimental rat model. The sequence of the gene coding for the large subunit of mitochondrial ribosomal RNA has been used to construct P. carinii specific oligonucleotide primers for the polymerase chain reaction. These oligonucleotides produced amplification of specific sequences from both P. carinii infected rat and human lung samplings, but none from a range of other organisms including potential pulmonary pathogens. Comparison of the sequence of amplified products from the infected rats and humans demonstrated limited but consistent differences between P. carinii from these two hosts and allowed for the construction of a human specific internal oligonucleotide. The application of the specific oligonucleotides for DNA amplification and subsequent Southern hybridisation affords extremely sensitive and specific detection of P. carinii in human samples, which ma3/be applicable to both epidemiological research and clinical studies. Key words: Pneumocystis carinii; DNA amplification
Introduction
Pneumocystis carinii is established as the prime cause of opportunistic pneumonia in patients with AIDS and those immunosuppressed on oncology and transplant units. Uncertainties about the parasite's taxonomy and the epidemiology of infection continue and are relevant to strategies for improving the prevention and treatment of this disease. The fastidious nature of P. carinii still demands the use of microscopy to identify the parasite in different samplings, after histochemical staining [1], with its inherent non-specificity or, more recently, immunofluorescence [2] of uncertain sensitivity [3]. There are sound theoretical grounds for believing that DNA amplification using the polymerase chain reaction (PCR) [4] might proCorrespondence address: Ann E. Wakefield, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, U.K. Abbreviations: PCR, polymerase chain reaction; bp, base pair.
vide both a highly specific and highly sensitive means of identifying the parasite. Materials and Methods
Cloning and sequencing of part of the gene coding for the large subunit of the mitochondrial ribosomal RNA from P. carinii. P. carinff pneumonia was induced in the rat model and DNA extracted and cloned from a parasite enriched fraction as previously described [5]. P. carinii specific sequences were confirmed by characteristic in situ " hybridisation patterns and recombinant plasmid pAZ102 was selected as a candidate mitochondrial sequence because of strong signals derived in dot blot hybridisation studies on infected samples. The recombinant plasmid pAZ102 (insert 570 bp) was sequenced using Sanger's chain termination method and the Sequenase kit (United States Biochemical Corporation, Cleveland, OH, U.S.A.), 35S (Amersham, U.K.), Sequagel (National Diagnostics, Manville, U.S.A.). The DNA sequence was compared with those available in
0166-6851/90/$03.50 © Elsevier Science Publishers B.V. (Biomedical Division)
70
Lanes- 1
2
3
4
5
6
7
8
9
(b)
(c)
• • e~
B~
71 Lan6s-
1
2
4
6
8
10
12
14
16
18 19 20
(b)
t
'----"--
•
Fig. 2. Results of DNA amplification using P. carinii specific oligonucleotide primers applied to DNA templates from P. carinii infected human lung and other organisms including potential pulmonary pathogens. Products of DNA amplification, using DNA templates prepared from: lane 2, rat; lane 4, human; lane 6, Candida albicans; lane 8, Candida (non-albicans); lane 10, Cryptococcus neoformans; lane 12, Mycobacterium tuberculosis; lane 14, Saccharomyces cerevisiae; lane 16, Aspergillus nidulans; lane 18, human-derived P. carinii. Lane 19, no template control; lanes 1 and 20: 1-kb molecular weight markers. (A) Ethidium bromide stained gel. (B) Autoradiograph of Southern blot probed with pAZI02-L2. Asterisks mark arrows pointing to bands of amplified DNA specific to P. carinii.
Fig. 1. Amplified DNA sequences demonstrated by ethidium bromide staining in pulmonary samplings from both rat and human hosts. Comparative oligoblotting with rat and human P. carinii specific internal oligonucleotide. Products of DNA amplification using DNA templates prepared from: lanes 2, 3, 4, rat-derived P. carinii; lanes 5, 6, 7, human-derived P. carinii. Lane 8, no template control; lanes 1 and 9, 1-kb molecular weight markers. (A) Ethidium bromide stained gel. (B) Autoradiograph of Southern blot probed with pAZI02-LI; (C) Autoradiograph of Southern blot probed with pAZ102-L2. Asterisks mark arrows pointing to bands of amplified DNA specific to P. carinii.
72 several databases including EMBL and GenBank. From the sequence data on pAZ102 and comparative analysis of the databases, the fragment was identified as a portion of the gene coding for the large subunit of the mitochondrial, ribosomal RNA of P. carinii and this showed significant homology with fungal sequences (manuscript in preparation).
Oligonucleotide primers. Two sequences of moderate conservation that were specific to P. carinii were selected for construction of oligonucleotide primers for the polymerase chain reaction: pAZ 102-E: -5'-GATGGCTGTTTCCAAGCCCA3'; pAZI02-H:-5'-GTGTACGTTGCAAAGTACTC-3'. An oligonucleotide for confirmatory Southern hybridisation on amplification products was chosen, pAZ102-L1. Subsequently a new internal oligonucleotide specific to human P. carinii sequences was constructed, pAZ102-L2 (Fig. 5). Template DNA. (i) Samplings for DNA amplification using our oligonucleotide primers comprised (a) pulmonary lavage samplings from 3 humans and 3 rats with P. carinii pneumonia documented by methenamine silver staining and microscopy, and (b) isolates from a series of organisms including some potential pulmonary pathogens: Candida (an albicans and a non-albicans strain), Cryptococcus neoformans, Mycobacterium tuberculosis, Saccharomyces cerevisiae and Aspergillus nidulans (Fig. 2). (ii) Template DNA was prepared from each sample by proteinase K digestion in the presence of SDS and EDTA followed by phenol/chloroform/ether extraction, and ethanol precipitation. DNA amplification. Using primers pAZ102-E and pAZ102-H the template samples, together with control samples without template, underwent 40 cycles of amplification performed with denaturation at 94°C for 90 s, annealing at 50°C for 90 s and extension at 72°C for 2 min (Techne, U.K.). The DNA amplification reaction mixture (50 11,1) contained 50 mM KC1/10 mM Tris, pH 8/0.01% (w/v) gelatin/3 mM MgC12/400 #M dNTPs (Boehringer Mannheim, U.K.)/0.4-1.0 #M
oligonucleotide/3 units of Amplitaq (Perkin Elmer Cetus, U.K.). To avoid the possibility of false negative results in the human clinical samples, i.e., failure to detect the specific amplification product for technical reasons, we carried out a parallel polymerase chain reaction on each sample using primers derived from the human anti-thrombin gene, exon 2 (S.L. Thein, personal communication). These primers, (ATI:-5'GTTGCAGCCTAGCTTAACTTGGCA- 3'; AT4:5'-GGT/'GAGGAATCATTGGACTTG-3') allowed amplification to take place using human genomic DNA as template. In each of the clinical samples the 500-bp specific product was detected, demonstrating efficient amplification. The potential problem of contamination in PCR was monitored by systematic use of the following techniques: (i) including several negative control samples with no added template DNA; (ii) by the use of UV irradiation of the PCR reaction mixes prior to the addition of the template DNA [6]; (iii) the use of separate disposable microcapillaries for the addition of each template (Laser Laboratory Systems Ltd., U.K.). The control samples remained negative in all experiments. Amplified products (10 #1) were electrophoresed in 1.5% agarose gels and visualised after ethidium bromide staining by ultraviolet light. The gel was Southern blotted on to Hybond N (Amersham, U.K.) and hybridised with 32p end-labelled internal primer at 46°C (pAZI02-L1) or 40°C (pAZ102-L2) for 3 h [7]. Filters were subsequently washed at high stringency at 54°C (pAZ102-L1) or 48°C (pAZ102-L2) and filters exposed to radiographic film at - 8 0 ° C with intensifying screens. The expected amplification product was 355 bp long in the rat derived parasite, that from the human derived parasite being 9 bp shorter.
Sequencing of ppvducts of DNA amplification. The PCR product was gel-purified and recovered from the agarose gel using Geneclean (Bio 101, Inc). The purified DNA was heat-denatured and sequenced as described above using primers pAZ102-H or pAZ102-E at 20 pmol//~l.
73
Lanes- 1 2
4
6
8
10
12
17
19
(b)
O
Fig. 3. Sensitivity of detection of amplification products using ethidium bromide staining and oligoblotting. Products of amplification using serially diluted DNA template prepared from 2 ml bronchoalveolar lavage from a patient with confirmed P. carinii infection. Lane 2, undiluted; lane 4, diluted x 10; lane 6, diluted x 102; lane 8, diluted × 103; lane 10, diluted x 104; lane 12, diluted x 105. Lane 17, no template control; lanes 1 and 19, l-kb molecular weight markers. (A) Ethidium bromide stained gel. (B) Autoradiograph of Southern blot probed with pAZI02-L2. Asterisks mark arrows pointing to bands of amplified DNA specific to P. carinii. Results
The oligonucleotide primers derived from rat P. carinii produced efficient amplification of specific
sequence from both rat and h u m a n hosts, shown by ethidium bromide staining (Fig. 1) but none
from the range of other organisms including s o m e potential p u l m o n a r y pathogens (Fig. 2). The internal oligonucleotide, p A Z 1 0 2 - L 1 , derived from the rat P. carinii, produced strong hybridisation signals on Southern hybridisation with amplified products from the infected rat lungs, but
74
Lanes l
2
4
~iiF
~ii!i!i
6
8
10
12
14
16
18
20
22
24 25 2627 28
30
(a)
1,018~,
344'~' 298,~
(b)
Fig. 4. DNA amplification from a series of human samplings and oligoblotting with human derived P. carinii internal oligonucleotide pAZ102-L2. Products of DNA amplification using DNA template prepared from 2 ml bronchoalveolar lavage sampling: lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, patients with confirmed P. carinii infection; lanes 24, 25, 26, 27, 28, immunocompetent patients without P. carinii infection; lanes I, 22, 30, l-kb molecular weight markers. (A) Ethidium bromide stained gel. (B) Autoradiograph of Southern blot probed with pAZI02-L2. Asterisks mark arrows pointing to bands of amplified DNA specific to P. carinii.
weak signal, at high stringency, with the amplified product derived from human samples although these were visible on ethidium bromide staining (Fig. 1). Direct sequencing of the amplified products from each of the rat and human samples allowed comparison of their sequence and demon-
strated limited but consistent differences between the P. carinii DNA from these two hosts which included 5 base changes in the sequence of the internal oligonucleotide pAZ102-L1 (Fig. 5). An oligonucleotide specific to the human derived organisms was constructed, pAZ102-L2,
75
pAZI02-L2
5'
A A G G
A G A T
A
Fig. 5. Comparative sequence of oligonucleotides pAZI02-LI (rat P. carinii) and pAZI02-L2 (human P. carinii).
which showed strong hybridisation with the amplified product from human P. carinii and conversely showed weak hybridisation with the rat P. carinii amplified product (Fig. 1). It produced no hybridisation with the PCR products of the range of other organisms tested (Fig. 2). Studies using serial dilutions of human derived P. carinii template DNA indicated that the application of oligoblotting with pAZ102-L2 to amplified DNA products increased the sensitivity of detection by at least 100-fold over visualisation by ethidium bromide staining (Fig. 3). The P. carinii oligonucleotide primers successfully amplified specific PCR product from bronchoscopic alveolar lavage samplings from 10 HIVpositive individuals with pneumocystis pneumonia as documented by positive methenamine silver staining on the lavage samples (Fig. 4). Lavage samples from 5 immunocompetent subjects were studied as controls. These failed to show specific PCR product by ethidium bromide staining or oligoblotting.
Discussion We have characterised a portion of the gene coding for the large subunit of mitochondrial, ribosomal RNA from P. carinii and comparative analysis of this indicates significant homology with fungal sequences. This result accords with data we have on other mitochondrial genes (manuscript in preparation) and the observations of other groups on ribosomal RNA [8]. We have identified P. carinii specific sequences from which we have constructed oligonucleotide primers which allow efficient amplification of part of this ribosomal RNA gene from P. carinii infecting both rat and human hosts but not from a range of other organisms including some potential pulmonary pathogens. These results indicate the specificity of the amplified products to P. carinii, confirmed on Southern hybridisation with internal oligonucleotide pAZI02-LI derived from the rat
sequence and applied to the rat pulmonary samplings. Results from the human samplings suggested the likelihood of differences in sequenEe between the amplified products from the rat and human. This was confirmed by comparison of sequences which indicated limited, but consistent differences. This finding is highly relevant to any hopes of applying DNA amplification for specific sensitive detection of P. carinii in various samplings. We have shown that our oligonucleotide primers can be used to identify the presence of P. carinii in a number of human bronchoalveolar lavage samples. By using our second internal oligonucleotide, pAZ102-L2, which is specific to human P. carinii, the sensitivity of detection of amplified product is increased 100-fold. Thus amplified P. carinii specific DNA is detectable in lavage diluted × 105 from patients with documented P. carinii pneumonia. In contrast, we found no P. carinii DNA in lavage from immunocompetent subjects, thus questioning further the current dogma that opportunistic pneumonia results necessarily from reactivation of latent pulmonary infection by the parasite [9]. DNA amplification and subsequent oligoblotting offers a method that combines high specificity and sensitivity for the detection of the parasite. This should be immediately applicable to the studies now required to address the unresolved epidemiology of this infection and the potential exists for the technique to be developed, by suitable calibration and automation now available [ 10,11], for the accurate diagnostic work required for therapeutic studies.
Acknowledgements We thank Professor S.J.G. Semple for the HIV positive lavage samples; Dr. S.L. Thein and Dr. R. Olds for the human anti-thrombin primers; and Dr. D. Young for the Mycobacterium tuberculosis DNA. This work was supported by the Royal Society (AEW), the Medical Research Council (FJP) and the Wellcome Trust (SB and KS). The sequence of pAZ102 is subject to patent applications.
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