Real Time Quantitative PCR for Adenovirus Hexon DNA

Quantitative analysis of subgroup C adenovirus DNA from these cellular DNA preparations can now be performed using real-time PCR (8). To assess the presence of adenovirus subgroup C DNA, a sensitive real-time PCR assay was developed using adenovirus hexon-specific primers and a TaqMan probe. Primers were selected from a region that is highly conserved among subgroup C viruses, but is significantly divergent among other subgroups. Primers were modified from those originally described by Pring-Akerblom et al. (10) to facilitate amplification of a conserved region of the subgroup C adenovirus hexon gene (nucleotides 21005-21290 of adenovirus type 5 (Ad5), GeneBank accession number AY339865). Primer and TaqMan probe sequences are presented in Fig. 1A. This reaction produces a 285-bp PCR product, and, as expected, the primers are able to amplify all four of the subgroup C viruses (Ad1, Ad2, Ad5, and Ad6) but do not amplify representatives from the other subgroups (Fig. 1B) (see Note 10). Serial dilutions (from 5 x 107 to 1 copy) of Ad2 DNA are included in each run to generate a standard curve for quantitative assessment of donor adenovirus DNA. This assay is able to detect five copies of the adenovirus genome (see Note 11). The range of the assay allows quantitation over at least 7 orders of magnitude (Fig. 1C).

5" > 3"

Group

Prtmtr (F>

O

C

C

A

T

T

A

c

C

T

T

T

G

A

C

T

C

T

T

c

T

G

T

C

Ad1

c

Ad2

c

Ad 5

c

AdE

T

A

Ad 12

T

A

c

T

G

C

c

B

Ad7

T

C

T

G

c

A

B

Ad16

T

c

T

G

A

0

Ad4B

T

c

T

G

C

c

-

G

E

Ad4

T

c

C

C

F

Ad41

T

c

T

G

c

Fig. 1. (A) Nucleotide sequences of primers and TaqMan probe are to the hexon region of adenovirus. (B) The subgroup C viruses as well as representatives from each of the other human adenovirus subgroups were amplified by real-time PCR using the hexon primers, and the products were run on an ethidium bromide-stained 1.8% agarose gel. Numbers indicate the adenovirus serotype tested. M, marker; -, negative water control; +, Ad2-positive control DNA.

Fig. 1. (A) Nucleotide sequences of primers and TaqMan probe are to the hexon region of adenovirus. (B) The subgroup C viruses as well as representatives from each of the other human adenovirus subgroups were amplified by real-time PCR using the hexon primers, and the products were run on an ethidium bromide-stained 1.8% agarose gel. Numbers indicate the adenovirus serotype tested. M, marker; -, negative water control; +, Ad2-positive control DNA.

3.3.1. Preparation of Viral DNA Standards

In addition to your samples of interest, each PCR run will need to include a series of wells containing viral DNA standard dilutions (see Note 12).

1. Determine the concentration of standard Ad genomic DNA (Gibco) and convert to copy numbers (molecules) of viral DNA (see Note 13).

2. Prepare 10-fold serial dilutions of Ad2 genomic DNA, ranging from 5 x 107 copies/5 |L to 5 copies/5 |L in PCR-grade water containing 50 ng/mL yeast tRNA (Ambion).

3. Aliquot diluted standards and store at -20°C until PCR setup (see Note 14).

3.3.2. Preparation of Primers

Prepare 50 |M (100X) concentrations of each primer in PCR-grade water, divide into 50 |L aliquots, and store at -20°C (see Note 15).

1. Decontaminate the PCR work station by turning on a UV light for 15 min prior to setting up any PCR reaction (see Note 16).

2. Thaw lymphocyte lysate and adenovirus DNA standards on ice, mix thoroughly by vortexing, and distribute each sample (5 |L/well) into a 96-well iCycler PCR plate. Keep the plate on ice during the PCR setup. Samples are generally run in triplicate for both unknowns and standards. Run the five copies per well standard in replicates of five.

3. Prepare the PCR master mix just before use with the following final concentrations: Qiagen 1X PCR buffer, 2.35 mM MgCl2, 0.2 mM dNTPs, 0.5 |M of each primer, 2.5 U Qiagen Hotstart Taq polymerase, and 0.1 |M of probe. Use PCR-grade water to balance the total volume to 45 ||L per reaction. Mix thoroughly and place 45-|L aliquots into 96-well PCR plate (see Note 17).

4. Cover the PCR plate with iCycler optical tape and centrifuge briefly at 300g to ensure that none of the PCR reaction solution is stuck to the tape or the well walls.

5. The PCR plate is run in a Bio-Rad iCycler thermocycler at: 1 cycle of 95°C x 15 min, followed by 50 cycles of 95°C, 15 s, 55°C, 1 min.

3.3.4. Data Analysis

With known amounts of input copy number, the target gene can be quantified in the unknown samples. To quantify the results we included serial dilutions of purified Ad2 DNA in each PCR run. To make a correlation between the initial template concentration and the real-time detection curve, a point has to be determined where the fluorescence signal exceeds the average background signal. This point is referred to as the cycle threshold (CT) value (see Note 18). The CT is reported as a cycle number at this point and decreases linearly with

Fig. 2. (A) Purified Ad2 DNA was serially 10-fold diluted, and two replicates of each dilution (from 5 x 107 to 5 copies) were tested. The threshold cycle values of the standard dilution are plotted against the log10 of the starting copy number. The equation of the line gives a correlation coefficient higher than 0.990, and the slope of the line is greater than -3.8. (B) The fluorescence intensity collected in real time for each sample was plotted against the number of PCR cycles. The horizontal orange line indicates the fluorescence cycle threshold (CT) setting, which is set at 10 standard deviations above the baseline emission. RFU, relative fluorescent unit.

Fig. 2. (A) Purified Ad2 DNA was serially 10-fold diluted, and two replicates of each dilution (from 5 x 107 to 5 copies) were tested. The threshold cycle values of the standard dilution are plotted against the log10 of the starting copy number. The equation of the line gives a correlation coefficient higher than 0.990, and the slope of the line is greater than -3.8. (B) The fluorescence intensity collected in real time for each sample was plotted against the number of PCR cycles. The horizontal orange line indicates the fluorescence cycle threshold (CT) setting, which is set at 10 standard deviations above the baseline emission. RFU, relative fluorescent unit.

increasing input target quantity. By plotting CT values against the known input copy number, a standard curve is generated with linear range covering 7-8 log units (see Note 19). The CT value (or cycle number where the fluorescence of the unknown crosses threshold) of the unknown sample can then be correlated to the copy number of the standard with a corresponding CT value.

The final quantity of viral DNA must be reported relative to some biological standard such as cell number (see Fig. 2) or weight of tissue or volume of serum. For this reason, accurate input information, such as cell counts, is essential. Alternatively, one can normalize samples to an external reference gene (see Note 20).

3.3.5. Subgroup C Serotype Determination

The real-time PCR assay described above efficiently detects all subgroup C adenoviruses. Nonetheless, there are small differences in sequence of the PCR product among the four subgroup C serotypes (Ad1, 2, 5, and 6). Thus the dominant serotype present in a clinical sample can be determined by sequencing the PCR product (see Note 21).

1. Run the real-time PCR hexon products on a 1.8% agarose gel.

2. Cut out the PCR bands of interest using a fresh razor blade for each sample.

3. Purify the gel slice using a Qiagen gel extraction kit according to manufacturer's instructions.

4. Elute the purified DNA in sterile water and sequence using the forward real-time PCR hexon primer (see Note 22).

Virus serotype is determined by sequence comparison between the PCR product sequence and known nucleotide sequences for subgroup C adenoviruses published in GenBank by the National Center for Biotechnology Information (NCBI). Serotypes are identified by conserved nucleotide base changes (Fig. 3).

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    How to pcr for adenovirus?
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