Journal of Virological Methods, 38 (1992) 113-122 0 1992 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/92/$05.00
113
VIRMET 01336
Chemiluminescent microtiter method for detecting PCR amplified HIV-l DNA Kazuo Suzuki, Naoaki Okamoto,
Susan Watanabe and Tokio Kano
Biomedical Research Center, Olympus Corporation, East Setauket, NY, U.S.A. (Accepted 7 January
1992)
Summary
A rapid and sensitive method for detecting HIV-l DNA sequences amplified by polymerase chain reaction (PCR) is described. The method uses solution phase hybridization for rapid and efficient annealing between digoxigeninlabeled targets and biotinylated capture probes. Hybrids containing biotin are captured onto streptavidin coated microwells and all other PCR components are washed away, including spurious amplification products. The presence of the digoxigenin-labeled amplified HIV target is then detected by antidigoxigenin-alkaline phosphatase conjugates using the chemiluminescent substrate PPD. This approach maintains high specificity by nucleic acid dependent capture, and high sensitivity by efficient solution hybridization. The method is rapid (2 hours), and capable of detecting 10 HIV targets. HIV; PCR; Chemiluminescence; Microtiter; Solution hybridization
Introduction
Polymerase chain reaction (PCR) is a powerful and increasingly popular method for detecting relatively rare sequences. Applications include the detection of rare infectious agents such as HIV-l (Saiki et al., 1988; Rayfield et al., 1988; Taylor et al., 1988) and Hepatitis B virus (Larzul et al., 1988) and genetic markers for disease such as sickle cell anemia (Chehab et al., 1987) or HLA typing (Erlich, et al., 1986; Saiki et al., 1986; Bugawan et al., 1988). Correspondence to: K. Suzuki, Biomedical Research Center, Olympus Corp., 3 Technology Drive, East Setauket, NY 11733, USA.
114
Simple and sensitive detection methods are required to use this amplification technology in the clinical or research environment. Early detection methods involve a dot-blot format (Bos et al., 1987) or an oligo hybridization format (Rayfield et al., 1988). However, since these methods used radioactive probes to achieve sensitive detection, their drawbacks were the instability and hazards associated with radioactivity. More recent methods have used nonradioactive methods such as timeresolved fluorescence (Dahlen et al., 1991), chemiluminescence (Schmidt, 1991), fluorescence (Inouye and Hondo, 1990) or color (Keller et al., 1991) as substitutes for radioactivity. These approaches are good but use reagents which are unavailable commercially, such as europium or acridinium conjugates, or use relatively long hybridization times (4 h to overnight) to achieve sensitivity. The approach described here uses solution hybridization and commercially available reagents to produce an assay system which is rapid, sensitive, and accessible. Using this method, PCR reactions containing 10 HIV copies can be detected in 2 h.
Materials and Methods DNA extraction A human cell line, MOLT 4, was obtained from American Type Culture Collection (Rockville, MD). A plasmid, pBHl0, which contains HIV-l DNA, was received from AIDS Research and Reference Reagent Program (NIH; Bethesda, MD). DNA from the cell line and the plasmid was isolated using standard protocols (Sambrook et al., 1989). PCR amphyication
of the target
Four different amounts of plasmid pBH 10 HIV- 1 DNA (10, 102, 104, lo6 copies) were amplified in the presence of 2pg of MOLT 4 DNA. The PCR reactions contained 0.5 PM of the HIV-l primers, SK145 (AGT GGG GGG ACA TCA AGC AGC CAT GCA MT) and SK431 (TGC TAT GTC AGT TCC CCT TGG TTC TCT) (Perkin-Elmer-Cetus Inc.), 100 PM each of dATP, dCTP, dGTP, 10 PM of digoxigenin-dUTP (Boehringer Mannheim), 90 PM of dTTP, 2.5 units of Taq polymerase (Perkin-Elmer-Cetus Inc.) in 100 ~1 of 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgC12, and 0.00 1% gelatin. Thirty cycles of amplification were carried out on a Hybaid HB-TRl thermocycler (National Labnet). The parameters of the cycle were: denaturation for 20 s at 94°C primer annealing for 25 s at 55°C and extension from primer for 1.25 min at 72°C. Another set of primers, SK38 (ATA ATC CAC CTA TCC CAG TAG GAG AAA T) and SK39 (TTT GGT CCT TGT CTT ATG TCC AGA ATG C), was amplified by PCR using the conditions described above for SK145 and SK431.
115
PCR reactions were analyzed by 4% NuSieve GTG agarose gel (FMC) and visualized with ethidium bromide. The amplified bands from the PCR reactions containing originally lo4 and lo6 copies of HIV DNA were detected at 141 base pairs for SK145 and SK431, and at 114 base pairs for SK38 and SK39 relative to standard size markers from Hue111 digests of phi X 174 DNA (Bethesda Research Laboratory). Biotin probe preparation
The Biotinylated capture probe was prepared using PCR in the presence of one of the following biotin labeled deoxyribonucleotide triphosphate: biotin-7dATP (Bethesda Research Laboratory), biotin-14-dATP (Bethesda Research Laboratory), biotin-16-dUTP (Boehringer Mannheim), or biotin-21 -dUTP (Clontech). Biotinylated probes were produced by PCR using 100 ~1 reactions containing 1 x lo6 copies of pBH10 plasmid DNA, 0.5 PM of the primers, YT5 (GAG ACC ATC AAT GAG GAA GCT GC) and YT6 (ATC TGG CCT GGT GCA ATA GGC C), 100 PM dATP, dCTP, and dGTP, 50 PM of biotindUTP and dTTP, and 2.5 units of Tuq polymerase in the buffer described above. The YT5 and YT6 primers were complementary to sequences between the SK145 and SK431 primers. After 30 cycles using the thermocycler parameters described above, probes were purified in Bio-Gel A- 15 (Bio-Rad) as described previously (Sambrook et al., 1989). The biotin-labeled probe was analyzed in a 4% NuSieve GTG agarose gel with ethidium bromide stain. The biotin labeled probe was detected at 75 base pairs based upon the DNA size marker. The concentration of the probe was estimated at 2 ng/$, based on the comparison of intensity of the DNA size marker. Preparative
of coating of the microtiter-well
with streptavidin
Microlite 2 microtiter dishes (Dynatech Laboratories, Inc.) were coated with streptavidin (10 pg/ml in 10 mM acetate buffer (pH 5.4)) by incubating at 4°C for 16 h. Following this adsorption step, the plates were washed three times with TSE buffer (10 mM Tris-Cl (pH 7.6), 150 mM NaCl, 1 mM EDTA) and then blocked with 1% BSA (Sigma) in TSE buffer at 37°C for 1 h. Plates were stored at 4°C and were stable for at least a month. Hybridization
and detection
Digoxigenin-labeled HIV- 1 targets were prepared for denaturation and hybridization by pipetting 1 ~1 per PCR reaction into 100 ~1 of hybridization buffer (0.75 M NaClO.075 M sodium citrate (pH 7.0), 2 ng/lOO ~1 biotinylated capture probe). Solutions were heated at 96°C for 10 min to denature the DNA and then incubated at 68°C for 15 min to allow hybridization. Solutions were then transferred to the streptavidin coated microtiter wells and incubated for 1 h at 37°C. Unbound components were removed by washing 3 times with 42°C
116
wash buffer (0.1 x SSC, 0.1% polyvinylpyrrolidone, 0.01% Tween 20). Alkaline phosphatase conjugated digoxigenin antibodies (Boehringer Mannheim) diluted 1:lOOOin TSE buffer were added to the microtiter plates for 10 min at 37°C. Free conjugate was removed by washing six times with wash buffer. As a final step, 100 ~1 of the chemiluminescent substrate LumiPhos (Lumigen, Inc.) was added to each well. The plates were incubated at 37°C and then read using the Micro Light luminometer (Dynatech Laboratories, Inc.) for RLU (Relative Light Units). Results
The principle of this assay system is as follows. Target sequences are amplified and labeled with digoxigenin-dUTP through the polymerase chain reaction. Aliquots are removed and hybridized in solution with biotinylated capture probes which are complementary to sequences between the PCR primers. Following hybridization, the solution is added to a streptavidin coated microwell which captures biotinylated capture probes. All other components are removed by washing and then an alkaline phosphatase labeled antidigoxigenin antibody is added. If digoxigenin labeled targets are present, the alkaline phosphatase-antibody conjugates bind and generate luminescent product which can be measured by a luminometer. Optimization
of assay components
and conditions
Different commercially available biotinylated capture probe dUTP and dATP nucleotides were used to synthesize capture probes for comparison in the assay. The biotinylated nucleotides used were biotin-7dATP, biotin- 14-dATP, biotin- 16-dUTP, and biotin-2 1-dUTP. Each capture probe was tested with PCR reactions containing Molt 4 DNA plus 0, 102, 104, or lo6 initial HIV copies. The results are shown in Fig. 1A. Although each type of capture probe worked in the assay, the best sensitivities were achieved by probes made with biotin-14-dATP and biotin-16-dUTP. Probes made with biotin-16-dUTP were used further to determine the optimal probe concentration. Probe concentration was varied from 0.5 ng/lOO ~1 to 8 ng/ 100 ~1. The results are shown in Fig. 1B. Optimum performance was obtained in a fairly narrow range of 2 to 4 ng/lOO ~1 hybridization volume. Subsequent experiments used 2 ng/ 100 hybridization volume. Biotinylated
Temperature and ionic strength conditions during conditions hybridization were tested for their effects on the assay performance. As a first step, hybridization solutions were varied from 0.1 x SSC to 8 x SSC at 60°C and tested with PCR reactions containing Molt 4 DNA plus 0 or lo4 initial HIV copies. The results are shown in Fig. 1C. Optimal performance was achieved in a broad range between 2.5 x SSC and 6 x SSC. 5 x SSC was
Hybridization
A
B 400
1000.
600. 200
2
,
400.
0 HIV
copy number
o
2
Concentration
4
6
01 probe
6
10
OX
2X
4X
6X
6X
(nglreaction)
Fig. 1. Optimization of probe hybridization conditions. Optimal conditions were determined using PCR reactions containing Molt 4 DNA plus 0, 10’ 104, or lo6 HIV prior to amplification. Biotin-16-dUTP, biotin-21-dUTP, biotin-7-dATP, and biotin-1CdATP were used to synthesize capture probes which were tested for relative performance (A). Capture probe made with Biotin-16-dUTP was used at 0.5 ng, 1 ng, 2 ng, 4 ng, 6 ng or 8 ng per 100 ~1 to determine the optimal concentration (B). Biotin-16-dUTP capture probe at 2 ng per 100 ~1 was tested in hybridization buffers of 0.1 x SSC, 1 x SSC, 2.5 x SSC, 5 x SSC, 6 x SSC, and 8 x SSC (C).
chosen for further studies to determine the optimal temperature. Hybridization temperatures of 60°C 68°C and 76°C were compared using PCR reactions containing Molt 4 DNA plus 0, IO*, 104, or IO6 initial HIV copies. The results are shown in Table 1. Similar signal and background levels were seen at all three temperatures, indicating a broad range of acceptable hybridization temperatures. Subsequent experiments used 68°C. Time for biotin-streptavidin capture The effect of time on the attachment of the biotinylated capture probes to streptavidin-coated microtiter plates was tested. Incubation times tested were 15, 30, 60, 90, and 120 min. Each time was evaluated using PCR reactions containing Molt 4 DNA and 0, lo*, 104, or lo6 TABLE 1 Optimal hybridization
temperature
for the detection
of HIV
HIV per PCRa
60”Cb
68”Cb
76”Cb
0 lo2 IO4 IO6
3.60 40.84 262.41 1,ooo.oo
3.08 40.68 288.66 1,ooo.oo
3.09 40.65 269.40 678.71
a Initial HIV copies used for PCR amplification. b Values are relative light unit (RLU) measured by Dynatech
luminometer.
6 1200
r’
800.
3 u
copies copies
-o-
1,ooo.ooo
4
10.000
-a-
100copes
-a- 0 copy
600-
400-
200-
0
0
20 40 Time
60
80
100 120
00
20
(min)
40
60
Time
(min)
80
100
Fig. 2. Optimization of signal detection. Time-course for capture of hybrids on streptavidin coated microtiter wells was tested (A). Time-course for generation of chemiluminescent signal was measured (B). PCR reactions used for comparison contained MOLT 4 DNA plus 0, IO’, 104, or lo6 HIV prior to amplification.
initial HIV copies. The results are shown in Fig. 2A. Capture reached a plateau in 60 min.
was rapid and
Incubation
time for alkaline phosphatase and substrate Time between addition of the chemiluminescent substrate and detection were varied as shown in Fig. 2B. Incubation times were evaluated using PCR reactions containing Molt 4 DNA and 0, lo’, 104, or lo6 initial HIV copies. Time to maximal signal, approximately 30 to 60 min, was similar for each PCR reaction.
TABLE 2 Compilation
of chemiluminescence
HIV per PCRa 0
IO2 IO4 lo6
signals from three assay sets
N
Meanb
*SD
cv
16 14 14 10
3.40 37.35 290.17 980.36
0.37 1.76 24.43 50.65
10.9 4.7 8.4 5.2
a Initial HIV copies used for PCR amplification. b Values are relative light unit (RLU) measured by Dynatech
luminometer.
119
-0
lOOO-
0
10
SK%-SK39
100
10,000
HIV copy number
1,000,000
10 100
10,000
HIV copy number
J
1,000,000
Fig. 3. Detection of HIV DNA. Signal levels were compared between amplified DNA which was complementary or noncomplementary to the biotinylated capture probe. (A). Primers SK38 and SK39 were used to amplify a noncomplementary region. SK145 and SK431 were used to amplify the complementary region. The mean and standard deviation from Table 2 were used to plot a standard curve (C). The lower limit of detection was set as indicated by the arrow.
Detection of HIV DNA Reproducibility The interassay variation was tested by repeat assays on different days using the optimized assay conditions described above. Each assay used the same set of PCR reactions containing Molt 4 DNA and 0, 102, 104, or lo6 initial HIV copies. The means and standard deviations for assays done on 3 days are shown in Table 2. Combining the results from three days yielded coefficients of variation of 5 to 10%. Specificity In order to check the specificity of the assay, PCR reactions containing amplified sequences noncomplementary (SK38 and SK39) to the capture probe were compared to PCR reactions containing complementary sequen:es (SK145 and X431). Both sets of primers were used to amplify 0, 10 , 10 , or lo6 HIV copies in the presence of Molt 4 DNA. The results are shown in Fig. 3A. Good specificity was maintained even in the presence of high levels of noncomplementary digoxigenin labeled DNA. Sensitivity A standard curve of initial HIV copies per PCR reaction versus signal (Fig. 3B) was constructed using the values from Table 2. Based on a calculation of the mean for the negative control (3.40) plus 6 x standard
120
deviations (6 x 0.37) a cutoff of 5.6 RLU (Relative Light Units) was obtained. This cutoff is indicates an analytical sensitivity equivalent to approximately 10 initial HIV targets per PCR reaction.
Discussion The polymerase chain reaction has expanded the applications of DNA probes into fields that were previously unattainable because of the requirement for high sensitivity (reviewed by Erlich et al., 1991; Persing, 1991). Other amplification methods such as Q-beta replicase (Lomell et al., 1989) and 3SR (Kwoh et al., 1989) although not as developed at the current time, may eventually also be applied to the detection of rare nucleic acid targets. A common problem to all the amplification techniques is the variation in accuracy between different laboratories. A recent example of the variation is seen in the multicenter PCR proficiency trial, which indicated that while sensitivity was high, specificity varied from 90 to 100% (Shepperd et al., 1991). In general, problems with specificity were due to spread of amplified target primarily at the PCR reaction step or at the detection step. Anti-contamination procedures and reagents have lowered false positives at the PCR reaction step, but they are not designed to improve accuracy at the detection step. Interestingly, a preliminary study has indicated that 66% of the false negatives occur at the detection step and not at the PCR reaction step (Madej et al., 1991). For this reason, development of technologies which help to improve the accuracy of detection should help to improve the performance of amplification technologies in general. Several detection methods have been reported. Initial formats involved gel electrophoresis combined either with ethidium bromide or 32P-labeled probes to visualize amplified products (Saiki et al., 1988; Kellogg and Kwok, 1990; Kwok et al., 1987). Ethidium bromide is relatively insensitive while solution probes depends on probe quality and hybridization with 32P-labeled electrophoresis conditions. More recent formats have used a basic ‘sandwich’ assay format wherein the amplified target DNA is captured onto a solid phase and then detected with nonisotopic signal systems such as color, time-resolved fluorescence, or chemically triggered chemiluminescence (Keller at al., 1991; Dahlen et al., 1991; Schmidt, 1991). The format described here is also a form of ‘sandwich’ assay but differs in that it uses commercially available reagents to produce a system which maintains high sensitivity. In addition, since alkaline phosphatase is used, fluorescent and calorimetric as well as chemiluminescent signal systems are available. Less sensitivity will be likely for the calorimetric signal system (Urdea et al., 1988). The sensitivity using calorimetric substrate for this assay has not been determined yet, but is likely to be approximately 10 times less sensitive than the current assay format. The components and conditions for this assay are generally robust and dependable. The exception appears to be the
121
narrow optimum concentration for the capture probe. This narrow tolerance may be due to limiting amounts of streptavidin molecules in the microtiter wells which are being exceeded with higher capture probe concentrations. This sandwich assay has a lower limit of detection at approximately 10 HIV DNA copies per PCR reaction. Estimates for the number of HIV targets in clinical specimens have been made using DQ alpha coamplification (Lee et al., 1991) PCR dilution (Simmonds et al., 1990) or culture (Ho et al., 1989). In general, these studies found that individuals with AIDS or ARC had relatively high HIV target numbers and were easily detectable. The same was true for most asymptomatic individuals; however, a small group of asymptomatic individuals were estimated to have levels as low as l-10 HIV DNA copies/lo5 PBMC’s. This low number is at the limit of detection for all reported detection systems and may require amplification using nested primers or HIV transcript plus HIV genome amplification. Amplification technology has provided a tool for direct study of infectious or inheritable factors. Detection of the amplified products requires simple, sensitive, and accessible methods. Until complete systems are available, researchers and clinicians will need to depend on assembling systems from commercially available components. The system described here can be used for any target of known sequence, is easily assembled, and has been optimized to provide sensitive and specific performance.
References of human Albert, J. and Fenyo, E.M. (1990) Simple, sensitive, and specific detection immunodeticiency virus type 1 in clinical specimens by polymerase chain reaction with nested primers. J. Clin. Microbial. 28, 156@1564. Bos, J.L., Fearon, E.F., Hamilton, S.R., Vries, M.V., Boom, J.H. and Vogelstein, B. (1987) Prevalence of ras gene mutations in human colorectal cancers. Nature 327, 293-297. Bugawan, T.L., Saiki, R.K., Levenson, C.H., Watson, R.M. and Erlich, H.A. (1988) The use of nonradioactive oligonucleotide probes to analyze enzymatically amplified DNA for prenatal diagnosis and forensic HLA typing. Bio/Technology 6, 943-947. Chehab, F., Cai, S.P., Doherty, M., Kan, Y.W., Cooper, S. and Rubin, E.M. (1987) Detection of sickle cell anemia and thalassaemisas. Nature 329, 293-294. Dahlen, P.O., Iitai, A.J., Skagius, G., Frostell, A., Nunn, M.F. and Kwiatkowski, M. (1991) Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescence-based hybridization assay. J. Clin. Microbial. 29, 798-804. Erlich, H.A., Sheldon, E.L. and Horn, G. (1986) HLA typing using DNA probe. Bio/Technol. 4, 975-981. Erlich, H.A., Gelfand, D. and Sninsky, J.J. (1991) Recent advances in the polymerase chain reaction. Science 252, 1643-1651. Ho, D.D., Moudgil, T. and Alam, M. (1989) Quantification of human immunodeticiency virus type 1 in the blood of infected persons. New Engl. J. Med. 321, 1621-1625. Inouye, S. and Hondo, R. (1990) Microplate hybridization of amplified viral DNA segment. J. Clin Microbial. 28, 1469-1472. Keller, G.H., Huang, D.P. and Manak, M.M. (1991) Detection of Human Immunodeticiency virus type 1 DNA by polymerase chain reaction amplification and capture hybridization in microtiter wells. J. Clin Microbial. 29, 6388641.
122 Kellogg, D.E. and Kwok, S. (1990) PCR protocols: a guide to methods and applications, Academic Press, San Diego, CA, pp. 337-347. Kwoh, D.Y., Davis, G.R., Whitfield, K.M., Chappelle, H.L., DiMichele, L.J. and Gingeras, T.R. (1989) Transcription-based amplification system and detection of amplified human immunodeticiency virus type 1 with a basd-based sandwich hybridization. Proc. Natl. Acad. Sci. USA 89, 1173-l 177. Kwok, S., Mack, D.H., Mullis, K.B., Poiesz, B., Ehrhch, G., Blair, D., Friedman-Kien, A. and Sninsky, J.J. (1987) Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer vleavage detection. J. Virol. 61, 169(X1694. Larzul, D., Giugue, F., Sninsky, J.J., Mack, D.H., Brechot, C. and Guesdon, J.L. (1988) Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J. Virol. Methods 20, 2277237. Lee, T., Suzeri, F.J., Tobler, L.H., Williams, B.G. and Busch, M.P. (1991) Quantitative assessment of HIV-I DNA load by coamplification of HIV-I gag and HLA-DQ-alpha genes. AIDS 5,6833 691. Lomell, H., Tyagi, S., Pritchard, C.G., Lizardi, P.M. and Kramer, F.R. (1989) Quantitative assay based on the use of replicatable hybridization probes. Clin. Chem. 35, 18261831. Madej, R., Louie, P., Rosa, C.D. and Rodgers, G. (1991) Analysis of HIV detection by PCR: Identification and reduction. Clin. Chem. 37, 1058. Persing, D.H. (1991) Minireview: polymerase chain reaction: trenches to benches. J. Clin. Micro. 29, 1281-1285. Rayfield, M., Cock, K.D., Heyward, W., Golgstain, L., Kwok, S., Lee, S., McCormick, J. and Ou, C. (1988) Mixed human immunodeficiency virus (HIV) infection in an individual. J. Infect. Dis. 158, 1170-1175. Saiki, R.K., Bugawan, T.L., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1986) Analysis of enzymatically amplified P-globin and HLA-DQa DNA with allele-specific oligonucleotide probes. Nature 324, 163-166. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487491. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press. Schmidt, B.L. (1991) A rapid chemiluminescence detection method for PCR-amplified HIV-l DNA. J. Virol. Methods 32, 233-244. Sheppard, H.W., Ascher, M.S., Busch, M.P., Sohmer, P.R., Stanley, M., Lute, J.C., Chimera, J.A., Madej, R., Rodgers, G.C., Lynch, D., Khayan-Bashi, H., Murphy, E.L., Eble, B., Bradford, W.Z., Royce, R.A. and Winkelstein, W. (1991) A multicenter proficiency trial of gene amplification (PCR) for the detection of HIV-l. J. Acquired Immune Detic. Syndr. 4, 277-283. Simmonds, P., Balfe, P., Peutherer, J.F., Ludlam, C.A., Bishop, J.O. and Brown A.J.L. (1990) Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J. Virol. 64, 864-872. Taylor, G.R., Hyde, K., Wensley, R.T. and Delamore, I.W. (1988) Polymerase chain reaction amplification and detection of HIV DNA sequences in the peripheral blood. Br. J. Haematol. 69, 127-133. Urdea, M., Warner, B.D., Running, J.A., Stempien, M., Clyne, J. and Horn, D. (1988) Comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res. 16, 49374956.
113
VIRMET 01336
Chemiluminescent microtiter method for detecting PCR amplified HIV-l DNA Kazuo Suzuki, Naoaki Okamoto,
Susan Watanabe and Tokio Kano
Biomedical Research Center, Olympus Corporation, East Setauket, NY, U.S.A. (Accepted 7 January
1992)
Summary
A rapid and sensitive method for detecting HIV-l DNA sequences amplified by polymerase chain reaction (PCR) is described. The method uses solution phase hybridization for rapid and efficient annealing between digoxigeninlabeled targets and biotinylated capture probes. Hybrids containing biotin are captured onto streptavidin coated microwells and all other PCR components are washed away, including spurious amplification products. The presence of the digoxigenin-labeled amplified HIV target is then detected by antidigoxigenin-alkaline phosphatase conjugates using the chemiluminescent substrate PPD. This approach maintains high specificity by nucleic acid dependent capture, and high sensitivity by efficient solution hybridization. The method is rapid (2 hours), and capable of detecting 10 HIV targets. HIV; PCR; Chemiluminescence; Microtiter; Solution hybridization
Introduction
Polymerase chain reaction (PCR) is a powerful and increasingly popular method for detecting relatively rare sequences. Applications include the detection of rare infectious agents such as HIV-l (Saiki et al., 1988; Rayfield et al., 1988; Taylor et al., 1988) and Hepatitis B virus (Larzul et al., 1988) and genetic markers for disease such as sickle cell anemia (Chehab et al., 1987) or HLA typing (Erlich, et al., 1986; Saiki et al., 1986; Bugawan et al., 1988). Correspondence to: K. Suzuki, Biomedical Research Center, Olympus Corp., 3 Technology Drive, East Setauket, NY 11733, USA.
114
Simple and sensitive detection methods are required to use this amplification technology in the clinical or research environment. Early detection methods involve a dot-blot format (Bos et al., 1987) or an oligo hybridization format (Rayfield et al., 1988). However, since these methods used radioactive probes to achieve sensitive detection, their drawbacks were the instability and hazards associated with radioactivity. More recent methods have used nonradioactive methods such as timeresolved fluorescence (Dahlen et al., 1991), chemiluminescence (Schmidt, 1991), fluorescence (Inouye and Hondo, 1990) or color (Keller et al., 1991) as substitutes for radioactivity. These approaches are good but use reagents which are unavailable commercially, such as europium or acridinium conjugates, or use relatively long hybridization times (4 h to overnight) to achieve sensitivity. The approach described here uses solution hybridization and commercially available reagents to produce an assay system which is rapid, sensitive, and accessible. Using this method, PCR reactions containing 10 HIV copies can be detected in 2 h.
Materials and Methods DNA extraction A human cell line, MOLT 4, was obtained from American Type Culture Collection (Rockville, MD). A plasmid, pBHl0, which contains HIV-l DNA, was received from AIDS Research and Reference Reagent Program (NIH; Bethesda, MD). DNA from the cell line and the plasmid was isolated using standard protocols (Sambrook et al., 1989). PCR amphyication
of the target
Four different amounts of plasmid pBH 10 HIV- 1 DNA (10, 102, 104, lo6 copies) were amplified in the presence of 2pg of MOLT 4 DNA. The PCR reactions contained 0.5 PM of the HIV-l primers, SK145 (AGT GGG GGG ACA TCA AGC AGC CAT GCA MT) and SK431 (TGC TAT GTC AGT TCC CCT TGG TTC TCT) (Perkin-Elmer-Cetus Inc.), 100 PM each of dATP, dCTP, dGTP, 10 PM of digoxigenin-dUTP (Boehringer Mannheim), 90 PM of dTTP, 2.5 units of Taq polymerase (Perkin-Elmer-Cetus Inc.) in 100 ~1 of 10 mM Tris-Cl (pH 8.3), 50 mM KCl, 1.5 mM MgC12, and 0.00 1% gelatin. Thirty cycles of amplification were carried out on a Hybaid HB-TRl thermocycler (National Labnet). The parameters of the cycle were: denaturation for 20 s at 94°C primer annealing for 25 s at 55°C and extension from primer for 1.25 min at 72°C. Another set of primers, SK38 (ATA ATC CAC CTA TCC CAG TAG GAG AAA T) and SK39 (TTT GGT CCT TGT CTT ATG TCC AGA ATG C), was amplified by PCR using the conditions described above for SK145 and SK431.
115
PCR reactions were analyzed by 4% NuSieve GTG agarose gel (FMC) and visualized with ethidium bromide. The amplified bands from the PCR reactions containing originally lo4 and lo6 copies of HIV DNA were detected at 141 base pairs for SK145 and SK431, and at 114 base pairs for SK38 and SK39 relative to standard size markers from Hue111 digests of phi X 174 DNA (Bethesda Research Laboratory). Biotin probe preparation
The Biotinylated capture probe was prepared using PCR in the presence of one of the following biotin labeled deoxyribonucleotide triphosphate: biotin-7dATP (Bethesda Research Laboratory), biotin-14-dATP (Bethesda Research Laboratory), biotin-16-dUTP (Boehringer Mannheim), or biotin-21 -dUTP (Clontech). Biotinylated probes were produced by PCR using 100 ~1 reactions containing 1 x lo6 copies of pBH10 plasmid DNA, 0.5 PM of the primers, YT5 (GAG ACC ATC AAT GAG GAA GCT GC) and YT6 (ATC TGG CCT GGT GCA ATA GGC C), 100 PM dATP, dCTP, and dGTP, 50 PM of biotindUTP and dTTP, and 2.5 units of Tuq polymerase in the buffer described above. The YT5 and YT6 primers were complementary to sequences between the SK145 and SK431 primers. After 30 cycles using the thermocycler parameters described above, probes were purified in Bio-Gel A- 15 (Bio-Rad) as described previously (Sambrook et al., 1989). The biotin-labeled probe was analyzed in a 4% NuSieve GTG agarose gel with ethidium bromide stain. The biotin labeled probe was detected at 75 base pairs based upon the DNA size marker. The concentration of the probe was estimated at 2 ng/$, based on the comparison of intensity of the DNA size marker. Preparative
of coating of the microtiter-well
with streptavidin
Microlite 2 microtiter dishes (Dynatech Laboratories, Inc.) were coated with streptavidin (10 pg/ml in 10 mM acetate buffer (pH 5.4)) by incubating at 4°C for 16 h. Following this adsorption step, the plates were washed three times with TSE buffer (10 mM Tris-Cl (pH 7.6), 150 mM NaCl, 1 mM EDTA) and then blocked with 1% BSA (Sigma) in TSE buffer at 37°C for 1 h. Plates were stored at 4°C and were stable for at least a month. Hybridization
and detection
Digoxigenin-labeled HIV- 1 targets were prepared for denaturation and hybridization by pipetting 1 ~1 per PCR reaction into 100 ~1 of hybridization buffer (0.75 M NaClO.075 M sodium citrate (pH 7.0), 2 ng/lOO ~1 biotinylated capture probe). Solutions were heated at 96°C for 10 min to denature the DNA and then incubated at 68°C for 15 min to allow hybridization. Solutions were then transferred to the streptavidin coated microtiter wells and incubated for 1 h at 37°C. Unbound components were removed by washing 3 times with 42°C
116
wash buffer (0.1 x SSC, 0.1% polyvinylpyrrolidone, 0.01% Tween 20). Alkaline phosphatase conjugated digoxigenin antibodies (Boehringer Mannheim) diluted 1:lOOOin TSE buffer were added to the microtiter plates for 10 min at 37°C. Free conjugate was removed by washing six times with wash buffer. As a final step, 100 ~1 of the chemiluminescent substrate LumiPhos (Lumigen, Inc.) was added to each well. The plates were incubated at 37°C and then read using the Micro Light luminometer (Dynatech Laboratories, Inc.) for RLU (Relative Light Units). Results
The principle of this assay system is as follows. Target sequences are amplified and labeled with digoxigenin-dUTP through the polymerase chain reaction. Aliquots are removed and hybridized in solution with biotinylated capture probes which are complementary to sequences between the PCR primers. Following hybridization, the solution is added to a streptavidin coated microwell which captures biotinylated capture probes. All other components are removed by washing and then an alkaline phosphatase labeled antidigoxigenin antibody is added. If digoxigenin labeled targets are present, the alkaline phosphatase-antibody conjugates bind and generate luminescent product which can be measured by a luminometer. Optimization
of assay components
and conditions
Different commercially available biotinylated capture probe dUTP and dATP nucleotides were used to synthesize capture probes for comparison in the assay. The biotinylated nucleotides used were biotin-7dATP, biotin- 14-dATP, biotin- 16-dUTP, and biotin-2 1-dUTP. Each capture probe was tested with PCR reactions containing Molt 4 DNA plus 0, 102, 104, or lo6 initial HIV copies. The results are shown in Fig. 1A. Although each type of capture probe worked in the assay, the best sensitivities were achieved by probes made with biotin-14-dATP and biotin-16-dUTP. Probes made with biotin-16-dUTP were used further to determine the optimal probe concentration. Probe concentration was varied from 0.5 ng/lOO ~1 to 8 ng/ 100 ~1. The results are shown in Fig. 1B. Optimum performance was obtained in a fairly narrow range of 2 to 4 ng/lOO ~1 hybridization volume. Subsequent experiments used 2 ng/ 100 hybridization volume. Biotinylated
Temperature and ionic strength conditions during conditions hybridization were tested for their effects on the assay performance. As a first step, hybridization solutions were varied from 0.1 x SSC to 8 x SSC at 60°C and tested with PCR reactions containing Molt 4 DNA plus 0 or lo4 initial HIV copies. The results are shown in Fig. 1C. Optimal performance was achieved in a broad range between 2.5 x SSC and 6 x SSC. 5 x SSC was
Hybridization
A
B 400
1000.
600. 200
2
,
400.
0 HIV
copy number
o
2
Concentration
4
6
01 probe
6
10
OX
2X
4X
6X
6X
(nglreaction)
Fig. 1. Optimization of probe hybridization conditions. Optimal conditions were determined using PCR reactions containing Molt 4 DNA plus 0, 10’ 104, or lo6 HIV prior to amplification. Biotin-16-dUTP, biotin-21-dUTP, biotin-7-dATP, and biotin-1CdATP were used to synthesize capture probes which were tested for relative performance (A). Capture probe made with Biotin-16-dUTP was used at 0.5 ng, 1 ng, 2 ng, 4 ng, 6 ng or 8 ng per 100 ~1 to determine the optimal concentration (B). Biotin-16-dUTP capture probe at 2 ng per 100 ~1 was tested in hybridization buffers of 0.1 x SSC, 1 x SSC, 2.5 x SSC, 5 x SSC, 6 x SSC, and 8 x SSC (C).
chosen for further studies to determine the optimal temperature. Hybridization temperatures of 60°C 68°C and 76°C were compared using PCR reactions containing Molt 4 DNA plus 0, IO*, 104, or IO6 initial HIV copies. The results are shown in Table 1. Similar signal and background levels were seen at all three temperatures, indicating a broad range of acceptable hybridization temperatures. Subsequent experiments used 68°C. Time for biotin-streptavidin capture The effect of time on the attachment of the biotinylated capture probes to streptavidin-coated microtiter plates was tested. Incubation times tested were 15, 30, 60, 90, and 120 min. Each time was evaluated using PCR reactions containing Molt 4 DNA and 0, lo*, 104, or lo6 TABLE 1 Optimal hybridization
temperature
for the detection
of HIV
HIV per PCRa
60”Cb
68”Cb
76”Cb
0 lo2 IO4 IO6
3.60 40.84 262.41 1,ooo.oo
3.08 40.68 288.66 1,ooo.oo
3.09 40.65 269.40 678.71
a Initial HIV copies used for PCR amplification. b Values are relative light unit (RLU) measured by Dynatech
luminometer.
6 1200
r’
800.
3 u
copies copies
-o-
1,ooo.ooo
4
10.000
-a-
100copes
-a- 0 copy
600-
400-
200-
0
0
20 40 Time
60
80
100 120
00
20
(min)
40
60
Time
(min)
80
100
Fig. 2. Optimization of signal detection. Time-course for capture of hybrids on streptavidin coated microtiter wells was tested (A). Time-course for generation of chemiluminescent signal was measured (B). PCR reactions used for comparison contained MOLT 4 DNA plus 0, IO’, 104, or lo6 HIV prior to amplification.
initial HIV copies. The results are shown in Fig. 2A. Capture reached a plateau in 60 min.
was rapid and
Incubation
time for alkaline phosphatase and substrate Time between addition of the chemiluminescent substrate and detection were varied as shown in Fig. 2B. Incubation times were evaluated using PCR reactions containing Molt 4 DNA and 0, lo’, 104, or lo6 initial HIV copies. Time to maximal signal, approximately 30 to 60 min, was similar for each PCR reaction.
TABLE 2 Compilation
of chemiluminescence
HIV per PCRa 0
IO2 IO4 lo6
signals from three assay sets
N
Meanb
*SD
cv
16 14 14 10
3.40 37.35 290.17 980.36
0.37 1.76 24.43 50.65
10.9 4.7 8.4 5.2
a Initial HIV copies used for PCR amplification. b Values are relative light unit (RLU) measured by Dynatech
luminometer.
119
-0
lOOO-
0
10
SK%-SK39
100
10,000
HIV copy number
1,000,000
10 100
10,000
HIV copy number
J
1,000,000
Fig. 3. Detection of HIV DNA. Signal levels were compared between amplified DNA which was complementary or noncomplementary to the biotinylated capture probe. (A). Primers SK38 and SK39 were used to amplify a noncomplementary region. SK145 and SK431 were used to amplify the complementary region. The mean and standard deviation from Table 2 were used to plot a standard curve (C). The lower limit of detection was set as indicated by the arrow.
Detection of HIV DNA Reproducibility The interassay variation was tested by repeat assays on different days using the optimized assay conditions described above. Each assay used the same set of PCR reactions containing Molt 4 DNA and 0, 102, 104, or lo6 initial HIV copies. The means and standard deviations for assays done on 3 days are shown in Table 2. Combining the results from three days yielded coefficients of variation of 5 to 10%. Specificity In order to check the specificity of the assay, PCR reactions containing amplified sequences noncomplementary (SK38 and SK39) to the capture probe were compared to PCR reactions containing complementary sequen:es (SK145 and X431). Both sets of primers were used to amplify 0, 10 , 10 , or lo6 HIV copies in the presence of Molt 4 DNA. The results are shown in Fig. 3A. Good specificity was maintained even in the presence of high levels of noncomplementary digoxigenin labeled DNA. Sensitivity A standard curve of initial HIV copies per PCR reaction versus signal (Fig. 3B) was constructed using the values from Table 2. Based on a calculation of the mean for the negative control (3.40) plus 6 x standard
120
deviations (6 x 0.37) a cutoff of 5.6 RLU (Relative Light Units) was obtained. This cutoff is indicates an analytical sensitivity equivalent to approximately 10 initial HIV targets per PCR reaction.
Discussion The polymerase chain reaction has expanded the applications of DNA probes into fields that were previously unattainable because of the requirement for high sensitivity (reviewed by Erlich et al., 1991; Persing, 1991). Other amplification methods such as Q-beta replicase (Lomell et al., 1989) and 3SR (Kwoh et al., 1989) although not as developed at the current time, may eventually also be applied to the detection of rare nucleic acid targets. A common problem to all the amplification techniques is the variation in accuracy between different laboratories. A recent example of the variation is seen in the multicenter PCR proficiency trial, which indicated that while sensitivity was high, specificity varied from 90 to 100% (Shepperd et al., 1991). In general, problems with specificity were due to spread of amplified target primarily at the PCR reaction step or at the detection step. Anti-contamination procedures and reagents have lowered false positives at the PCR reaction step, but they are not designed to improve accuracy at the detection step. Interestingly, a preliminary study has indicated that 66% of the false negatives occur at the detection step and not at the PCR reaction step (Madej et al., 1991). For this reason, development of technologies which help to improve the accuracy of detection should help to improve the performance of amplification technologies in general. Several detection methods have been reported. Initial formats involved gel electrophoresis combined either with ethidium bromide or 32P-labeled probes to visualize amplified products (Saiki et al., 1988; Kellogg and Kwok, 1990; Kwok et al., 1987). Ethidium bromide is relatively insensitive while solution probes depends on probe quality and hybridization with 32P-labeled electrophoresis conditions. More recent formats have used a basic ‘sandwich’ assay format wherein the amplified target DNA is captured onto a solid phase and then detected with nonisotopic signal systems such as color, time-resolved fluorescence, or chemically triggered chemiluminescence (Keller at al., 1991; Dahlen et al., 1991; Schmidt, 1991). The format described here is also a form of ‘sandwich’ assay but differs in that it uses commercially available reagents to produce a system which maintains high sensitivity. In addition, since alkaline phosphatase is used, fluorescent and calorimetric as well as chemiluminescent signal systems are available. Less sensitivity will be likely for the calorimetric signal system (Urdea et al., 1988). The sensitivity using calorimetric substrate for this assay has not been determined yet, but is likely to be approximately 10 times less sensitive than the current assay format. The components and conditions for this assay are generally robust and dependable. The exception appears to be the
121
narrow optimum concentration for the capture probe. This narrow tolerance may be due to limiting amounts of streptavidin molecules in the microtiter wells which are being exceeded with higher capture probe concentrations. This sandwich assay has a lower limit of detection at approximately 10 HIV DNA copies per PCR reaction. Estimates for the number of HIV targets in clinical specimens have been made using DQ alpha coamplification (Lee et al., 1991) PCR dilution (Simmonds et al., 1990) or culture (Ho et al., 1989). In general, these studies found that individuals with AIDS or ARC had relatively high HIV target numbers and were easily detectable. The same was true for most asymptomatic individuals; however, a small group of asymptomatic individuals were estimated to have levels as low as l-10 HIV DNA copies/lo5 PBMC’s. This low number is at the limit of detection for all reported detection systems and may require amplification using nested primers or HIV transcript plus HIV genome amplification. Amplification technology has provided a tool for direct study of infectious or inheritable factors. Detection of the amplified products requires simple, sensitive, and accessible methods. Until complete systems are available, researchers and clinicians will need to depend on assembling systems from commercially available components. The system described here can be used for any target of known sequence, is easily assembled, and has been optimized to provide sensitive and specific performance.
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122 Kellogg, D.E. and Kwok, S. (1990) PCR protocols: a guide to methods and applications, Academic Press, San Diego, CA, pp. 337-347. Kwoh, D.Y., Davis, G.R., Whitfield, K.M., Chappelle, H.L., DiMichele, L.J. and Gingeras, T.R. (1989) Transcription-based amplification system and detection of amplified human immunodeticiency virus type 1 with a basd-based sandwich hybridization. Proc. Natl. Acad. Sci. USA 89, 1173-l 177. Kwok, S., Mack, D.H., Mullis, K.B., Poiesz, B., Ehrhch, G., Blair, D., Friedman-Kien, A. and Sninsky, J.J. (1987) Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer vleavage detection. J. Virol. 61, 169(X1694. Larzul, D., Giugue, F., Sninsky, J.J., Mack, D.H., Brechot, C. and Guesdon, J.L. (1988) Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J. Virol. Methods 20, 2277237. Lee, T., Suzeri, F.J., Tobler, L.H., Williams, B.G. and Busch, M.P. (1991) Quantitative assessment of HIV-I DNA load by coamplification of HIV-I gag and HLA-DQ-alpha genes. AIDS 5,6833 691. Lomell, H., Tyagi, S., Pritchard, C.G., Lizardi, P.M. and Kramer, F.R. (1989) Quantitative assay based on the use of replicatable hybridization probes. Clin. Chem. 35, 18261831. Madej, R., Louie, P., Rosa, C.D. and Rodgers, G. (1991) Analysis of HIV detection by PCR: Identification and reduction. Clin. Chem. 37, 1058. Persing, D.H. (1991) Minireview: polymerase chain reaction: trenches to benches. J. Clin. Micro. 29, 1281-1285. Rayfield, M., Cock, K.D., Heyward, W., Golgstain, L., Kwok, S., Lee, S., McCormick, J. and Ou, C. (1988) Mixed human immunodeficiency virus (HIV) infection in an individual. J. Infect. Dis. 158, 1170-1175. Saiki, R.K., Bugawan, T.L., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1986) Analysis of enzymatically amplified P-globin and HLA-DQa DNA with allele-specific oligonucleotide probes. Nature 324, 163-166. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B. and Erlich, H.A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487491. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press. Schmidt, B.L. (1991) A rapid chemiluminescence detection method for PCR-amplified HIV-l DNA. J. Virol. Methods 32, 233-244. Sheppard, H.W., Ascher, M.S., Busch, M.P., Sohmer, P.R., Stanley, M., Lute, J.C., Chimera, J.A., Madej, R., Rodgers, G.C., Lynch, D., Khayan-Bashi, H., Murphy, E.L., Eble, B., Bradford, W.Z., Royce, R.A. and Winkelstein, W. (1991) A multicenter proficiency trial of gene amplification (PCR) for the detection of HIV-l. J. Acquired Immune Detic. Syndr. 4, 277-283. Simmonds, P., Balfe, P., Peutherer, J.F., Ludlam, C.A., Bishop, J.O. and Brown A.J.L. (1990) Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J. Virol. 64, 864-872. Taylor, G.R., Hyde, K., Wensley, R.T. and Delamore, I.W. (1988) Polymerase chain reaction amplification and detection of HIV DNA sequences in the peripheral blood. Br. J. Haematol. 69, 127-133. Urdea, M., Warner, B.D., Running, J.A., Stempien, M., Clyne, J. and Horn, D. (1988) Comparison of non-radioisotopic hybridization assay methods using fluorescent, chemiluminescent and enzyme labeled synthetic oligodeoxyribonucleotide probes. Nucleic Acids Res. 16, 49374956.
