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Q uantification of proviral P N A load bv end-point dilution

The use of nested PCR allows the detection of a single target copy added to the reaction (Siramonds et al. 1990). Therefore by amplification of multiple replicates from a dilution series of target DNA or cDNA it is possible to estimate the copy numbers in a given volume of the original sample. The method used in this thesis is based on the method of Simmonds et al. (1990) in which the sample is diluted in a five-fold dilution series in replicate until only a proportion of the replicates give a positive signal. At this end-point the number of copies from which the positive reactions aie obtained will follow a Poisson distribution (i.e. some reactions will come from a single target copy, some from two etc.). The frequency of negative replicates (F) allows the calculation of copy numbers at this sample dilution as :

copies/unit volume = - In [F]

In practice four replicates were amplified from a five-fold dilution series (neat to 1:125) using the pol primer set by the method described in Section 2.1. This strategy covered the majority of samples from HIV-1 infected subjects and those in which all replicates gave a positive reaction at a dilution of 1:125 were repeated, diluting from

1:125 to 1:15625.

Q uantification of cell-free virus RNA

The method used for quantification of cell-free virus load by quantitative PCR was developed by Malcolm Semple in The Department of Virology, UCL (Semple et al. 1993). The assay is based on a nested PCR, using the pol primer set designed for diagnostic detection of HIV-1, in which a '^S labelled dNTP (^^S dCTP) was incorporated into the second round product and was quantified by scintillation counting. Labelled product was separated from unincorporated label by affinity capture of the product onto streptavidin coated micro titre wells via a biotinylated second round primer (POL 1-bio) incorporated into the second round product and, after washing away of unincorporated label, separation of the unbiotinylated strand by NaOH treatment for transfer to a scintillation cocktail for counting.

Test samples were quantified by comparison with an external standard curve run in the same assay. The standard curve was produced from a high titre patient sample previously quantified by end-point dilution PCR as described above.

RNA samples for assay were prepared from semm by affinity capture of virus paiticles as described in Section 2.3 and reverse transcribed as described in Section 2.3. Serum was preferred to plasma since intra-assay variability was lower with this material.

A separate antisense primer (CPI) immediately downstream of PCR primer MH6 was used in the reverse transcription step.

CPI 5' GGAGGGGTATTGACAA 3' (16 mer)

After removal of the latex by centrifugation, 10pi of the cDNA sample was added to a 50pl PCR reaction using pol primers MH 5 and MH 6 as detailed in Section 2.1. The first round reaction was run for 30 cycles using the conditions shown in Section 2.1 and Ip l of the first round product was transferred to the second round reaction mix shown below.

per tube

p/if polymerase 0.125pl

nucleotide mix (GAT mix) 0.4pl

lOx reaction buffer 2.5pl

primer 1 (POL 1-bio) 1.25 pi

primer 2 (POL 2) 1.25pi

''S dCTP Ip l

sample or control Ip l

Thermal cycling conditions were as follows:

0 0 0

12 cycles 94 C for 1min., 50 C for 1 min., 75 C for 1 min.

0

1 cycle 75 C for 7 mins.

0

1 cycle 4 C indefinitely

In the second round of amplification pfu polymerase (Recombinant exo-; Stratagene Ltd., Cambridge, U.K.) was used in preference to taq polymerase as the incorporation of dNTPs by taq is poor. This enzyme requires a higher primer

0

concentration, as shown above, and has an optimum extension temperature of 75 C. The nucleotide mix contains G,A and T only as the C is provided by the label, and the mix was used at 1/10 normal amount to balance with the nucleotide concentiation of the label.

The second round PGR was run for 12 cycles as this was found to give the widest dynamic range of quantification whilst still detecting a single cDNA input copy. All samples and conti'ols were purified, reverse transcribed and amplified in duplicate. Each duplicate was assayed in duplicate giving four values for each sample or control.

From each second round product 5ql was mixed in duplicate with 95ql PBS, containing 0.05% Tween 20, in a strep ta vidin coated micro titre well. Coating was as described in Section 2.5, except that the wells were coated with lOOql/well rather than 25ql. The wells were incubated at room temperature for one hour and washed 10 times with TTA buffer with a one minute soak between washes six and seven. After aspirating the wash buffer, 60ql 0.15M NaOH was added to each well, incubated at room temperature for five minutes, 50ql of the NaOH solution was transferred to a white microtitre plate (Canberra Packard) and mixed with lOOql scintillation cocktail (Microscint 40, Canberra Packard). Wells were counted for one minute in a Topcount microtitre scintillation counter (Canberra Packard), the mean of the replicates calculated, a standard curve plotted from the controls and the test samples read from this curve.

The standard curve was a half-log dilution series from 1000 cDNA copies to one cDNA copy made from a high titre patient serum diluted in negative human serum.