Effect of Nonionic Detergents on Amplification of Human Papillomavirus DNA with Consensus Primers MY09 and MY11

Crude cell lysates, obtained by protein digestion and membrane lysis with nonionic detergents such as Nonidet P-40 (NP-40), Tween 20, or Laureth 12, followed by heat denaturation, are suitable for PCR analysis (1). The L1 consensus PCR assay with the MY09-MY11 primer pair is widely utilized for detection of human papillomavirus (HPV) DNA (2, 3, 7, 10). However, the use of NP-40 is not proposed in PCR protocols using MY09-MY11 (1). Several investigators consider that NP-40 could impair the amplification process with MY09-MY11 (5a), although some have used it with success (4–6, 9). The effect of NP-40 on amplification with MY09-MY11 has not been reported yet. Since NP-40 is not inhibitory for Taq polymerase at concentrations below 0.1% (8) and since it is commonly used in sample-processing protocols, we have evaluated the effect of NP-40 on the detection of HPV DNA with the MY09-MY11 primer pair.

Ten-microliter volumes of the detergent preparations described below were added to 90-μl volumes of a PCR master mix containing the usual components (2, 7) plus eight copies of HPV-16 (kindly provided by H. zur Hausen), 5 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Montréal, Canada), and the L1 consensus HPV primers MY09 and MY11. Detergent solutions included (i) 10 μl of Tris-HCl (pH 7.4) with 0.01 mM EDTA (TE) (no-detergent control solution), (ii) 10 μl of a solution of 0.4% Tween 20 and 0.4% NP-40, (iii) 10 μl of a solution of 0.8% Tween 20, (iv) 10 μl of a solution of 0.8% NP-40, and (v) 10 μl of a solution of 0.8% Laureth 12 (kindly provided by P. Gravitt, Roche Molecular Systems). Final concentrations of nonionic detergents in the amplification mixtures reached 0.08%. This corresponds to the final concentration of detergents in the PCR mixture, since in our usual PCR protocols specimens are treated with 0.8% detergents and represent 10% of the final amplification reaction volume. Amplification was performed in a model 9600 thermal cycler with the following cycling conditions: activation of AmpliTaq Gold at 95°C for 9 min; 40 cycles of DNA amplification at 95°C for 20 s, 55°C for 30 s, and 72°C for 30 s; and a final elongation step at 72°C for 5 min. Amplified material was subjected to electrophoresis on a 2% agarose gel stained with ethidium bromide and visualized on a UV transilluminator.

As shown in Fig. 1, detection of eight copies of HPV-16 was impaired by the presence of NP-40 with or without Tween 20. Results similar to those with Tween 20 were obtained with Laureth 12 (data not shown). Three independent experiments generated similar results. The detection of 80 copies of HPV-16 DNA was not affected by the presence of NP-40 (data not shown). Our results do not support the use of NP-40 for cell lysis of samples analyzed with the MY09-MY11 primer pair for detection of low-viral-load infections. Tween 20 or Laureth 12 did not alter the efficiency of PCR amplification for HPV detection at concentrations of at most 0.08%.

FIG. 1.

Amplification of eight copies of HPV-16 accomplished in the presence of TE buffer (lanes b, c, and d), 0.04% (final concentration [vol/vol]) NP-40 and 0.04% Tween (lanes e and f), 0.08% Tween 20 (lanes g and h), and 0.08% NP-40 (lanes i and j). The negative control is in lane a. Amplification was done with AmpliTaq Gold DNA polymerase in a model 9600 DNA thermal cycler. A 450-bp band is visible in positive reactions.