JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1992, p. 2887-2892

Vol. 30, No. 11

0095-1137/92/112887-06$02.00/0

A Novel Polymerase Chain Reaction Method for Detection of Human Immunodeficiency Virus in Dried Blood Spots on Filter Paper JOSEPH YOURNO* AND JAMES CONROY Virology Laboratory, New York State Department of Health, Wadsworth Center for Laboratory and Research, Empire State Plaza, Albany, New York 12201-0509 Received 3 February 1992/Accepted 14 August 1992

A method for detection of proviral human immunodeficiency virus DNA in dried blood spots on filter paper by direct polymerase chain reaction (PCR) has been developed. To develop the method, a standard system was used which was prepared from cells each containing a single integrated provirus and titrated with normal donor blood. This rapid procedure provides virtually quantitative yields of nuclear DNA and exploits most of the standard methodology descxibed for blood specimens. A nested PCR using SK38-SK39 gag as the internal primer pair was also designed; this PCR detected a single copy of provirus per filter at near theoretical frequency with SK19 probe. The utility of the procedure was demonstrated with clinical specimens. Blood spot filters from human immunodeficiency virus-infected and uninfected individuals were readily and unequivocally discriminated. The method is designed for ultimate use with large (1.5-ml) sample preparation tubes that are compatible as PCR tubes with thermal cyclers. This will permit convenient, direct single-tube PCR of dried blood specimens on filters. It should be adaptable to analysis of dried blood spots for a variety of infectious or genetic diseases.

There is an urgent need for diagnostic tests that can reliably differentiate human immunodeficiency virus (HIV)infected from uninfected newborns for early detection and treatment (8, 14, 17). These tests must be highly sensitive as well as specific, since the viral load may be very low in a newborn (14). This need extends to studies based on dried blood or tissue specimens collected far afield from the testing site. Polymerase chain reaction (PCR) is increasingly being applied to this problem. Methods in current use are generally based on extraction and purification of target DNA with ionic detergents and organic solvents from blood spots on filter paper (4, 9, 11, 15). While capturing both chromosomal DNA and extrachromosomal DNA, these multistep methods suffer from inevitable loss of DNA template, increased chances for false positives through many open-tube manipulations, and episodic inhibition of PCR by the harsh organic reagents used for DNA extraction. This report describes a novel and efficient method for extraction of DNA from dried blood spots on filter paper for detection of HIV proviral sequences by PCR (18). The simple two-step procedure is designed to optimize the yield of high-quality DNA and to minimize manipulations for more reliable detection of target nuclear DNA sequences down to the single-copy level.

locytes, 64% mononuclear cells]; hematocrit, 45 to 50%) was used for threefold serial dilutions of 8E5 cells (starting count, 4,600 + 960 per ,ul). The 8E5 cell line is stably infected with HIV, each cell containing one copy of integrated HIV proviral DNA defective in the pol gene (5). From each dilution, 1/2-in. (1 in. = 2.54 cm) blood spots were prepared containing 33 to 0 8E5 cells (equal to provirus copies) per 1/4-in. filter circle, equivalent to 12 ,ul of blood containing 408,000 + 70,000 WBCs. The calculated amount of starting DNA per filter was 2.04 + 0.35 ,ug, based on 1 ,g of DNA per 200,000 WBCs. Blood spots were prepared dropwise (about 50 ,ul of blood) with a 1-ml serological pipette onto a standard newborn screening filter paper (DOH-1514; Schleicher & Schuell 903) to approximate spots from newborn heel punctures. Spots were allowed to dry for 2 to 4 h at room temperature, and the cards were stored in plastic bags at 4°C until used over a period of 24 months. A biosafety hood was used for all manipulations of open specimens up to PCR. Pipetting was done with plugged serological pipettes, microliter pipettes (Rainin) equipped with filter microliter pipette tips (USA Scientific Plastics) or positive displacement microliter pipettes (Rainin). Reagents for DNA extraction and PCR (see below) were prepared, aliquoted for single runs, and stored in a separate laboratory. Removal of hemoglobin. Filter circles from dried blood spots were prepared with a flamed 1/4-in. punch onto sterile paper, and then each was transferred with flamed forceps to a 1.5-ml gasketed screw-cap tube (Sarstedt). The filters were suspended in 1.25 ml of lysis buffer (0.32 M sucrose, 10 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 1% Triton X-100, all from Sigma) (2) and rotated at 36 rpm for 30 min at room temperature. The tubes were then microcentrifuged at 12,500 x g for 1 to 3 min, and the hemoglobin-tinged supernatants were carefully aspirated or decanted. This step was repeated once. The tube contents were allowed to drain upright for 1 to 2 min, and residual supernatant was aspirated. The residual volume with filter is about 30 ,ul. Processed filters were stored at -20°C.

MATERIALS AND METHODS Lysis method. The lysis method is adapted from the procedure of Higuchi (7). Hemoglobin is eliminated from the filter by cytoplasmic lysis (2) of erythrocytes. Leukocyte (WBC) nuclear DNA is left adherent to the filter. Digestion of the residual material on the filter to liberate DNA is done with the filter under constant agitation in PCR buffer with nonionic detergents. The digest with filter is subjected directly to PCR and analyzed. Blood spot materials. A normal donor buffy coat (American Red Cross) (WBCs, 34,000 + 5,800 per ,ul [36% granu*

Corresponding author. 2887

2888

YOURNO AND CONROY

DNA extraction from filters. Residual blood material on the processed filter and any pelleted material were digested at 37°C on a continuous-action vortexer at 1,000 rpm for 7 to 12 of 1.5 x PCR buffer (containing Tween 20 and h in 50 Nonidet P-40 [Sigma], both at 0.68%, and 100 ,ug of proteinase K [Boehringer-Mannheim] per ml) adjusted to 40 mM in Tris-HCl (pH 8.3) and 3.75 mM in MgCl2 (7). An aliquot of 8 of supematant (1/10 volume) was taken from the spun digest for DNA determination by fluorometry (6). Standard curves were established before each run with peripheral blood mononuclear cell (PBMC) DNA. The DNA aliquots in PCR buffer were each diluted in 2 ml of freshly prepared fluorometry buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl [pH 7.4], 0.1 ,ug of Hoechst 33258 dye per ml) and read with a Hoefer fluorometer. Digests were stored at -20°C for PCR. DNA recovery experiments. A fluorometric analysis of DNA recoverability from standard blood spot punches by this method was performed with a set of filters (0 copies of 8E5) digested for 0 to 16 h in duplicate. The kinetics of release of DNA into the supernatant in vortexed and stationary digests were compared. To determine residual DNA in the filter after the first digestion, the filter was resuspended in 50 ,l of fresh PCR buffer with proteinase K, teased apart with a micropipette tip, and reincubated stationary at 370C for 18 h (end or "limit" digest). In these experiments, a 25-pl aliquot of supernatant was taken from each spun digest for DNA determination. To determine the efficiency of extraction, DNA recovered from each filter after the first and limit digests was related to the starting DNA per filter, calculated as described above. PCR and analysis of product. An efficient nested PCR procedure which is capable of detecting reliably a single copy of target provirus allows increased diagnostic sensitivity for specimens with low-copy-number HIV. It also serves here as an independent check of DNA yield from filters. The nested PCR procedure for HIV gag sequences as devised here is based on using SK380 and SK390 as outer primers (see below), SK38 and SK39 as internal primers, and SK19 as probe (12). The digest supernatants with filter were boiled for 10 min and snap-cooled in ice water. The spun tube each) were transferred contents including filter (about 72 to a 0.5-ml Gene Amp reaction tube (Perkin-Elmer Cetus). PCR master mix and 4 to 7 drops of mineral oil were added. Negative controls consisted of distilled water and/or 1.3 ,ug of normal donor PBMC DNA, purified by the rapid procedure. Positive controls consisted of the above spiked with 60 to 100 provirus copies in DNA from Hut 78-HIV/aav, a cell line containing one integrated HIV provirus per genome (1). The cold PCR tubes were placed in a thermal cycler (PerkinElmer Cetus) set at a 94°C soak cycle, and cycling was started immediately. Both amplifications 1 and 2 consisted of 35 cycles, followed by an extension step. Final reaction mix parameters for each amplification were as follows: amplification 1, 102-pl volume, 2.5 mM MgC12, 25 pmol each of outer primers (high-pressure liquid chromatography [HPLC] purified; Operon) (SK380, 5' GAG AAC CAA GGG GAA GTG ACA TAG CAG G, 28-mer, gag 684-712 [HIV clone HXB2]; SK390, 5' TAG AAC CGG TCT ACA TAG TCT CTA AAG GG, 29-mer, gag 874-903 [HIV clone HXB2]), 5 U of AmpliTaq, 200 jiM each deoxynucleoside triphosphates; amplification 2, 140-jil volume, 2.5 mM MgCl2, 135 pmol each of inner primers SK38 and SK39 (HPLC purified; Operon) an additional 7 U of AmpliTaq, an additional 200 jiM each deoxynucleoside triphosphates (14). For amplification 2, 38 jil of PCR master mix 2 was added directly to the original PCR tube after completion of ampli-

J. CLIN. MICROBIOL.

fication 1. For both PCR amplifications 1 and 2 of the nested procedure, each cycle consisted of (i) ramp to 95°C, 75 s; (ii) 95°C, 20 s; (iii) ramp to 55°C, 75 s; (iv) 55°C, 30 s; (v) ramp to 60°C, 30 s; (vi) 60°C, 120 s. The extension step was 60°C, 10 min (1). A 10-,u aliquot of completed PCR amplification mix was hybridized with 250,000 cpm of SK19 probe 5' end labelled with 32p, at a final NaCl concentration of 150 mM. The hybridization mixture was boiled for 10 min and then incubated at 55°C for 30 to 90 min. An aliquot of hybridization mixture was analyzed by polyacrylamide gel electrophoresis (12). Kodak XAR-2 film was exposed to completed gels in cassettes equipped with intensifying screens. Standard conditions included S RI of hybridization mixture and 4 h of film exposure at room temperature. For greater signal strength, 12.5-,u aliquots were run and film was exposed for 4 h at -700C. Clinical specimens. Peripheral blood collected in EDTA was used to make 1/2-in. blood spots by pipetting 50 pl per spot onto standard newborn screening cards with a Rainin micropipette. The blood was allowed to dry for 2 h, and the cards were then stored at 4°C until use. Duplicate 1/4-in. blood spot filters from known infected and uninfected individuals (13 in each group) were used in nested PCR in a blind experiment. The uninfected group included 10 children (no. 1, 2, 5, 6, 7, 16, 18, 19, 24, and 25) of seropositive mothers from 3 days to 8 months of age who have been monitored to subsequent seroreversion (Table 1). These children had no laboratory (negative PCR and antigen testing) or clinical evidence of HIV infection and remain well. The sources of the blood spots from the other three uninfected individuals (no. 11, 13, and 26) were seronegative adults with no risk factors for HIV infection (Table 2). Blood spots from eight adult seropositive individuals (no. 4, 8, 9, 10, 17, 20, 21, and 22) who were intravenous drug users or sex partners of infected persons were tested (Table 2). Six of these individuals were asymptomatic, and two had neurologic symptoms. Each of the eight adults had positive HIV serology on more than one specimen and was PCR positive with both gag and env primer sets with 1 ,ug of mononuclear cell DNA. Blood spots from five infected pediatric patients (no. 3, 12, 14, 15, and 23) who ranged in age from 8 weeks to 7 years were also tested (Table 1). The infection status of these children was confirmed by positive PCR with mononuclear cell DNA. Antigenemia, persistence of seropositivity past 15 months, or development of HIV-related symptoms provided additional evidence of HIV infection in these children. All blood spots had been stored at 4°C for 24 to 30 months before the analysis was performed.

RESULTS Recovery of DNA from blood spot filters. While erythrocyte lysis released the hemoglobin from the filters into solution, the WBC DNA remained firmly bound to the paper. Digestion of filter material with vortexing released 75% or more of the calculated starting DNA into solution. The remainder could be detected in the paper residue (Fig. 1). The sum of DNA yields from the first and limit digestions approximated the calculated total starting DNA per filter, i.e., about 2 ,ug. The expected inverse relationship was evident between the curves of DNA released from the first and limit digests, i.e., the increase of supernatant DNA in the first digest was mirrored in an equivalent decrease in residual DNA in the filter. Agreement between duplicate digests was close except for with one pair of 2-h digests. Digestion of filter material

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TABLE 1. Laboratory and clinical assessment of children Filter DNAd (ng)

Child

Age

Serology by

PCR

CD4

CDC

Filter

no.a

(mo)

EIAIWB/Agb

gag/env

Total

PBMC

(no./mm3)

stage'

PCRf

3

29 29 379 83 86g 559 79 0.759 7 2.59 11 0.759 9 0.259 11 0.75 39 13 79 26 39 11 lg 14 19 15 3 89

+/+ +I+ +/+

683

498

515 150

416 96

315 409 397

221 160 233

683

498

ND ND 996 297 ND 501 1,381 ND ND ND ND ND ND ND ND ND ND ND

12 14

15 23 1

2 5 6 7 16

18 19

24 25

+/+/+ +I+I+

+/+/0

ND

+/+/ND +/+/ND

+/+ +/+ +/+ 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0 0/0

+/+/0 +/+/+ +/+/0 +/+/ND +/+/ND 0/ND/ND +/+/0 0/ND/0 +/+/0 0/ND/0

+/+/0 ND 0/ND/ND +/+/ND 0/ND/0

+/+/0 0/0/ND +/+/ND 0/ND/ND +/+/0 0/ND/ND +/+/0 0/ND/ND

318

283

349

182

467

377

478

383

1,600

506

380

ND ND ND

566

486

628

399

295

212

P1 P2 P2 P2 P2 P2 P2 P0 P0 P0

++++ ++++ ++++ ++++ ++++

0 0

Neg P0

0

Neg P0 Neg P0

0

P0 P0 P0

Neg P0 Neg P0 Neg P0 Neg P0 Neg

1,850 1,834 ND ND 1,687 1,354

0

0 0 0 0 0

The first 5 children listed make up the infected group; the other 10 make up the uninfected group. b EIA/WB/Ag, enzyme-linked immunosorbent assay/Western blot (immunoblot)/antigen. +, positive; 0, negative; ND, not done. c Results of PCR performed with 1 jig of mononuclear cell DNA with SK68-SK69 (env) and SK101-SK145 (gag) primer sets. d Maximum target DNA in blood spot ifiter calculated from hematology data. Total DNA = WBC per microliter x 12 (the blood volume in filter [microliters]) x (1 Lg of DNA per 200,000 WBC). PBMC DNA = total DNA x % lymphocytes and monocytes (from differential count). I CDC, Centers for Disease Control. P1, known infection, asymptomatic; P2, known infection, symptomatic; PO, unknown infection status; Neg, known absence of infection. f++ + +, strongly positive; 0, negative. g Source of specimen from which blood spot was prepared. a

without vortexing was less efficient, releasing about 50% of the DNA into solution in 8 h (Fig. 1). There was occasional slight to moderate interference apparent in fluorometry of small amounts of free DNA, probably due to nonionic

Adult no.a

Age (yr)

TABLE 2. Information on adult specimen bloodspots HIV serology by Filter DNAd (ng) PCR gag/envELISA/WBi or ELISA alone

4 8 9 10 17 20 21 22 11 13 26

26 32 38 61 29 40

38 37

detergents (6). This was manifested as occasional readings which were less than zero. Digests of paper blank filters gave values within the 0 0.1-,ug range (data not shown). These results indicate that virtually quantitative yields of template

+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ 0 0 0

+/+ +/+ +/+ +/+ +/+ +/+ +/+ +/+ 0/0 0/0 0/0

CDC' stage

Filter PCRf

Total

PBMC

200 97 235 279 341 148

126 50 54 36 259 101

II+++ II+++ II+++ IV ++++ II ++++ II ++++

254

155

II

455 454 239 395

178 236 69 138

IV

The first eight adults make up the infected group; the other three make up the uninfected group. b ELISA/WB, enzyme-linked immunosorbent assay/Western blot (immunoblot). Members of the uninfected group were tested c See Table 1, footnote c. d See Table 1, footnote d. I CDC, Centers for Disease Control. f See Table 1, footnotef. a

only by ELISA.

++++ ++++ 0 0 0

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YOURNO AND CONROY

2890

LANE No. 1 2 3

4 5 6 7 8 9 10 11 12 131415 16 17 18 19 20

t

t)

C.(H)

w

C.)

-i

C]

w _

-i

. U.

I

LL

INCUBATION TIME (h)

w cl w

0 2.5 w

1

3

11t

33

PROVIRUS COPIES

cc

2.0

z >

1.5

B) LIMIT DIGEST v

1.0

-

0.5

-

"

4

TXT

~SXTTX

>. +VXT -------

0.0 -0.5

o 1

2

4

6

8 12 18

PREVIOUS INCUBATION TIME (h) FIG. 1. Recovery of DNA from blood spot punches by rapid extraction procedure, as determined by fluorometry. (A) First digest. Symbols: 0, vortex (V) digestion; *, stationary (S) digestion; A, teased (T) (disrupted) punch control. Calculated starting DNA per punch, 2.04 + 0.32 jig. (B) Limit digest of once-digested punches. Symbols: 0, previous vortex digest (VXT); previous stationary digest (SXT); A, previously teased punch digest (TXT). The mean value of duplicate digests is given, while vertical lines show the deviation of duplicates. E,

DNA may be achieved by this procedure. In a separate experiment, DNA recoveries from filters taken from the center of the blood spot (1.78 0.25 ,ug, n = 5) and filters taken from the periphery (1.69 0.19 ,ug, n = 7) were not found to be significantly different. Released genomic DNA was examined by electrophoresis (lx Tris-borate-EDTA buffer, pH 8.3) in 0.8% agarose gels with ethidium bromide and was seen as a smear from high to low molecular weight. The released DNA tended to have a lower apparent molecular weight with digestion time, whether from vortexed or stationary digests (data not shown). Nevertheless, released DNA from vortexed digests appeared mainly to have an apparent molecular size of 1 kbp and was an excellent substrate for PCR (see below). PCR detection of HIV. The nested system proved extremely sensitive down to a single copy of provirus (Fig. 2 and Table 3). Similar results were obtained with duplicate filters prepared to contain 50 and 1,000 8E5 provirus copies in normal adult blood samples anticoagulated with EDTA (prepared as described for clinical specimens; data not shown). No false positives were found. Signal strength was uniformly strong down to the single-copy level, at which 1 moderate and 2 very weak signals, best detected after film exposure at -70°C (data not shown), were scored versus 11

FIG. 2. Detection of HIV SK38-SK39 PCR amplification product from the nested procedure by polyacrylamide gel electrophoresisautoradiography. A 5-,ul aliquot was analyzed, and X-ray film was exposed for 4 h at room temperature. Lanes: 1 to 4, 0 provirus copies; 5 to 14, 1 provirus copy; 15 and 16, 3 provirus copies; 17 and 18, 11 provirus copies; 19 and 20, 33 provirus copies. Composite of two gels to illustrate moderate single-copy positive (lane 11) and weak single-copy positive (lane 8). The sizes of oligonucleotides and the partially double-stranded hybrids are given on the vertical axis. The single-stranded SK19 probe is 41 nucleotides in length (41 ss). Hybrids consist of one or more strands of the SK38-SK39 amplification product (115 bp) base paired to SK19 probe. As deduced from these and numerous other experiments, the major hybrid bands seen here are heteroduplexes (115+41 het) and heteroquadruplexes [(3x) 115+41].

strong signals. Multiple hybrid forms corresponding to heteroduplexes and higher-order hybrids were seen in positive specimens. These results approach the theoretical limit of sensitivity as calculated for a cumulative Poisson distribution of 8E5 cells in the blood spot filter to the single cell level. They validate the high efficiency of target DNA recovery by the new method. Data are from four successive runs. In the first run only (six specimens), outer primers were reduced to 8 pmol each. No obvious differences were found. Standard, unnested PCR was also performed on filter digests with SK38 and SK39 primers. Final PCR reagent concentrations were as given for amplification 2 of the nested procedure (100-,u final volume), and an identical thermal cycler program was followed. This system detected between 1 and 3 provirus copies, but with a very weak to indeterminate signal below 11 copies when probed as described above with SK19 (data not shown). When tested by agarose gel electrophoresis and ethidium bromide fluorescence, all strong positives, as previously detected by hybridization and autoradiography, showed a prominent single SK38-SK39 amplification product band (data not shown). All negatives and the three weak to moderate single-provirus-copy positives showed no detectable SK38-SK39 product. The size of the band was as expected for the SK38-SK39 amplification product, 115 bp; its identity has previously been confirmed by Southern blots with SK19 probe (data not shown). Clinical specimens. The nested PCR was able readily and unequivocally to discriminate the blood spots from infected and uninfected individuals (Fig. 3). Both spots from each of the 13 infected individuals gave strong PCR products after amplification. At the same time, no detectable bands were found in duplicate testing of spots from negative individuals,

very

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TABLE 3. PCR detection of HIV provirus in spotted normal blood spiked with 8E5 cells: nested SK38-SK39 gag system Provirus copies per 1/4-in. filter (mean + SD)

No. tested

DNA recovery per punch (%)a

0 1.2 ± 0.3 3.6 ± 0.8 10.7±2.2 32.3 ± 6.7

20 20 12 4 4

84 15 74 + 18 79 + 7 75 5 57 13d

Positivesb

Signal strengthc

0/0 14/(10-16)

0 2 +w, 1 ++, 11 ++++ 12 ++++ 4++++ 4 ++++

12/(10-12) 4/4 4/4

Based on 2.04 ,ug of starting DNA per filter as a 100% value. Results are means + standard deviations. for 90% input for PCR after fluorometry. The first number in each pair is that observed; Based on Poisson distribution of 8E5 cells in 1/4-in. filter, the second number is that expected (95% confidence interval). I For positives, relative signal strength is rated qualitatively from +w (very weak) to + + + + (very strong). d Partial filters to approximate single 1/4-in. filter circles. a

corrected

b

the method suggest that the nested PCR will be useful in this application. The basic method should be adaptable to analysis of a variety of infections and genetic conditions and has several inherent advantages over those in current use. First, it avoids the harsh organics (e.g., sodium dodecyl sulfate, phenol, and chloroform) used in the standard procedures for extracting DNA (10). DNA liberated from PBMCs by the gentler rapid extraction procedure is considered a more dependable substrate for PCR amplification (13). Second, it allows virtually quantitative yields of nuclear DNA from the filter since most of the DNA remains with the paper after the only separation step (erythrocyte lysis). Third, it limits the number of purification steps, at which specimen preparation tubes must be opened, and thereby reduces the chances of false positives through contamination. It has been suggested that most of the DNA from cell digests is captured in the aqueous phase after extraction with phenol-chloroform but that significant amounts of DNA may be lost in each alcohol precipitation step (16). These factors and the frequent need for multiple or repeat steps make for significant loss of DNA template even under optimal conditions. Although direct calculations of DNA yield from blood spot filters cannot be made from data presented, published

even after 16 h of film exposure at room temperature. These conditions were shown to approximate the sensitivity of exposure at -70°C with these materials.

DISCUSSION A novel method for DNA extraction from blood spot filters for detection of HIV proviral DNA sequences by PCR has been developed. It is capable of detecting a single copy of proviral HIV DNA at near the frequency predicted by the cumulative Poisson distribution for 8E5 cells on the blood filter. Use of the nested PCR with blood spots from clinical specimens showed the assay to be both sensitive and specific. The nested assay is capable of unequivocally detecting infection in individuals when suboptimal amounts of mononuclear cell DNA are present. Typically, 1 to 3 jig of DNA is amplified in HIV PCR when nucleic acid is isolated from whole blood. The maximum amount of mononuclear cell DNA from infected individuals in the blood spot filters tested was less than 1 ±g in all cases. Although no blood spot DNA from infected newborns was amplified in these experiments, the near theoretical frequency of detection of one copy of proviral DNA in the standard system and the specificity of

A

B

1 2 3 4 5 6 7 8 9 101112131415161718

1 2 3 4 5 6 7 8

91011S1213141516171X81920

*= s~~

*:W

0 1 1 3 111 2 3 4 5 6 7 8 9 1011 1213

0 1 1 3 113333

8E5 Copies

8E5 Copies

Clinical Specimens

14151617l81920212223242526 Clinical Specimens

FIG. 3. Detection of nested PCR product from clinical specimens by hybridization and autoradiography. Amplification products were incubated with radiolabeled SK19 probe. After electrophoresis of the hybridization mixture, the gel was used to expose X-ray film for 4 h at room temperature. Arrowheads indicate hybrids formed between the SK38-SK39 amplification product and SK19 probe. See legend to Fig. 2 for further details. (A) Lanes 1 through 5 contain specimens from blood spots with 0, 1, 1, 3, and 11 copies of 8E5 proviral DNA, respectively. Clinical specimens 1 through 13 are in lanes 6 through 18. (B) Lanes 1 through 7 contain 0, 1, 1, 3, 11, 33, and 33 copies of 8E5 proviral DNA. Clinical specimens 14 through 26 are in lanes 8 through 20.

2892

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YOURNO AND CONROY

studies of normal donor materials using phenol-chloroform methods suggest the following yields: from direct digests of filters, about 0.8 ,ug of DNA per 75 RI of dried blood (21 to 42% yield) (4); from direct digestion of methanol-fixed filters, about 0.15 to 0.50 ,g/12 RI of dried blood (24 to 167% yield) (9); from digests of saline eluates of shredded or minced filters, about 0.5 ,g/50 ,lI (11) and 2.0 ,g/200 RI of dried blood (15) (21 to 42% yield). Calculations of approximate yield are based on 5,000 to 10,000 WBCs per pI of blood and 1 pg of DNA per 200,000 WBCs. A recent study suggests that PBMCs of HIV-infected adults may harbor extrachromosomal viral cDNA in quiescent lymphocytes as well as chromosomal provirus in cells which have undergone full proviral integration. Extrachromosomal viral cDNA may account for 50% or more of total HIV DNA in early infections (3). Since this method was developed with nuclear DNA from blood spot cells containing integrated provirus, any cytoplasmic viral cDNA could be lost at the lysis step. However, integrated forms which would be detected along with nuclear extrachromosomal cDNA are present from early infection (3). Furthermore, since integration of viral cDNA to produce provirus is the hallmark of established retroviral infection, whereas preintegration in quiescent cells may not result in true infection of these cells (3), the detection of proviral DNA as described herein is the ultimate criterion of infection. HIV-positive specimens generated two or more major bands on autoradiography after hybridization with SK19 probe, but a single SK38-SK39 product band of 115 bp was visualized by agarose gel electrophoresis and ethidium bromide fluorescence. This is the expected size of the doublestranded SK38-SK39 amplification product before hybridization to SK19 probe. The standard unnested PCR amplification with internal primers SK38 and SK39 also generated the same pattern of bands by each technique (data not shown). Hence, the multiple hybrid bands seen on autoradiography do not reflect loss of primer specificity in PCR amplification 2 but represent heteroduplexes and more complex reassociations of amplification product and probe. This PCR method for blood spot filters has been developed for ultimate use with (1.5-ml) specimen preparation tubes that can also serve as PCR tubes. This would allow the entire process from treatment of filters through amplification to be done with single tubes for optimum simplicity, reliability, and sensitivity. ACKNOWLEDGMENTS The methodology was developed by Joseph Yourno, with the blood spot standard system. James Conroy contributed the clinical portion of this study, including clinical laboratory studies and patient materials, as well as certain other reagents. Malcolm Baker of James Conroy's laboratory performed the hybridization experiments with amplified clinical specimens. The following people from the Virology Laboratory also contributed materials or facilities to this project: Evelyn Keller, 8E5 cells, and Leo Grady, space for preparation and storage of PCR reagents. Consultation on PCR with Alessandra Sardelli, Perkin-Elmer Cetus, Wilton, Conn., is also acknowledged. A. Sardelli, Chin-Yeh Ou, Centers for Disease Control, Atlanta, Ga., and Anne Comeau, Massachusetts State Laboratory, Jamaica Plain, Mass., provided a critical reading of the manuscript before submission. The dedicated typing assistance of Margaret Prescott is also acknowledged.

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