Quantification of

Enterovirus RNA in

Sludge Samples Using

Single Tube Real-Time

RT-PCR

BioTechniques 29:88-93 (July 2000)

ABSTRACT

We have developed a quantitative RTPCR method that can be used to determine

the amount of enterovirus RNA in urban

sludge samples. This method combines TaqManÒ technology with the ABI PrismÔ

7700 real-time sequence detection system.

We optimized a one-step RT-PCR that uses

a dual-labeled fluorogenic probe to quantify

the 5¢ noncoding region of enteroviruses.

For accurate quantification of the number

of copies, a Mahoney type 1 poliovirus RNA

standard was designed and produced using

genetic engineering. This fragment, quantified using the RibogreenÒ method, was used

in serial dilutions as an external standard.

The method had a 7-log dynamic range (5

to 2  ́ 107). PCR inhibitors were removed

by extracting viral RNA (after virus concentration) using the RNeasyÒ mini kit with

added polyvinylpyrrolidone (PVP) and running the amplification reaction with a mixture containing PVP and T4 gene 32 protein. This real-time quantification of

enterovirus RNA allows large numbers of

samples to be screened. Its sensitivity, simplicity and reproducibility render it suitable

as a screening method with which to characterize enteroviruses, the presence of infectious particles being subsequently confirmed by cell culture.

INTRODUCTION

There are generally two kinds of

methods for detecting enteric viruses in

environmental samples, depending on

whether they are based on cell culture

infectivity or on molecular detection

methods such as PCR followed by nucleic acid hybridization (16). Compared

to time-consuming cell culture assays,

PCR is rapid, sensitive and specific. It is

also capable of detecting non-cytopathogenic, slow-growing and rare viruses

in complex environmental samples such

as sewage or sludge (20). Current amplification techniques are either qualitative (10) or quantitative with the measurement of the amount of product

amplified as their end point (25).

In this study, we suggest a singletube, one-step, real-time RT-PCR assay

for the quantification of enteroviruses

in sludge samples using TaqMan

technology (Roche Molecular Biochemicals, Meylan, France). This technology makes use of the 5′→3′ nucleolytic activity of AmpliTaq DNA

polymerase and uses primer pairs in

conventional amplification and a duallabeled fluorogenic probe (5,13). Three

major difficulties need to be overcome

in the development of such a technique:

(i) because enteroviruses are RNA

viruses, RT-PCR needs to be optimized

in a single step to keep manipulation

time to a minimum; (ii) because treated

sludge contains only small quantities of

enterovirus, a highly sensitive technique is required; and (iii) the purest

possible genetic material needs to be

extracted because sludge samples contain numerous organic compounds

(polyphenols and humic acids) and

heavy metals that are liable to inhibit

amplification enzymes (7,17,21).

The use of polyvinylpyrrolidone

(PVP) in genome extraction and the T4

gene 32 protein during amplification

enabled us to achieve a complete protocol, from the extraction of virus from

sludge to the counting of genome

copies. Here, we present the first results

obtained with samples of natural and

artificially contaminated sludge. We

compare these results with measurements made using cell culture, which

remains the reference technique.

MATERIALS AND METHODS

Residual Sludge

Three types of sludge were sampled

from two waste-water treatment plants

in Lorraine, France. Primary sludge

was taken from the primary decanting

ponds of the Nancy and Metz treatment

plants; activated sludge and thickened

sludge from a secondary decanting

pond were taken from the Metz plant.

Samples of thickened sludge (78% dry

solids) loaded with 7 × 104 and 7 × 103

pfu poliovirus 1 vaccine (Sabin) were

used as controls.

Virus Extraction and Concentration

Technique

Virus was eluted from sludge samples using the technique described by

Ahmed and Sorensen (1). A volume of

sludge providing 10 g (dry solids), to

which 100 mL 10% beef extract (pH

9.0) (Oxoid, Basingstoke, UK) had

been added, was stirred at 500 rpm for

30 min. The mixture was sonicated on

ice (100 W, 0.9 s) for 5 min in 1-min

steps, mixed again for 5 min and then

centrifuged at 5000× g for 1 h at 4°C.

The extract consisted of the supernatant,

neutralized to pH 7.2. This extract was

concentrated using polyethylene glycol

(PEG) 6000 (Prolabo, Fontenay sous

bois, France) and precipitated according

to the Lewis and Metcalf technique

(12). An 8% final concentration (w/v)

PEG 6000 (in PBS, pH 7.2, bioMerieux, Lyon, France) was added to

the extract. After vigorous agitation, the

mixture was stored overnight at 4°C and

then centrifuged at 10 000× g for 90 min

at 4°C. The pellet was resuspended in

10 mL PBS. Finally, this concentrate

was decontaminated by adding a onethird volume of chloroform (10-min

centrifugation at 1500× g) followed by

200 μL antibiotic cocktail (penicillin

100 000 IU/mL, colimycin 250 000 IU/

mL, streptomycin 100 mg/mL and amphotericin B 0.5 mg/mL).

Quantification of Infectious

Enterovirus Using Cell Culture

Quantities of the present infectious

enterovirus were measured by inoculating the decontaminated concentrates in vitro into buffalo green

monkey cell cultures in 96-well microplates. All cultures were inoculated

in duplicate using 40 wells for each dilution (fivefold serial dilutions). Each

well was filled with 50 μL inoculum

and 200-μL nutritive medium [Eagle

minimum essential medium (Eurobio,

Les Ulis, France), to which 5% newborn calf serum had been added] containing 1.5 × 105 cells/mL. The cultures were incubated at 37°C in 5%

CO2

for five days. Virus density was

Short Technical Reports

88 BioTechniques Vol. 29, No. 1 (2000)
determined from the cytopathic effects

observed after duplicate inoculation of

cell layers with three successive dilutions of the sample. The cytopathic effect was confirmed by two serial passages. The mean viral concentration

for each sample was estimated using

the most probable number (MPN)

method with software devised by Maul

(15). Results were expressed in the

most probable number of colony unit

(MPNCU)/mL sample and then converted to MPNCU/10 g sludge (dry

solids) to take into account any variations in sludge dryness.

Extraction of Viral RNA

Enterovirus RNA was extracted

from 250 μL concentrate using the

RNeasy plant mini kit (Qiagen,

Courtaboeuf, France) according to the

manufacturer’s instructions, with the

following exception: the kit RLT buffer

was modified by the addition of 2%

(w/v) PVP 40 000 (Sigma, St. Quentin,

France) (4,22,26).

Fluorogenic RT-PCR

To design the primers and probe for

the TaqMan technology, the most

constant genome region in enteroviruses, the 5′-noncoding region (5′ NCR),

was chosen. The software Primer Express was used and identified primer

Ev1 [5′-GATTGTCACCATAAGCAGC-3′] (position 579–597), primer Ev2

[5′-CCCCTGAATGCGGCTAATC-3′]

(position 451–469) and Ev-probe [5′-

FAM-CGGAACCGACTACTTTGGGTGTCCGT-TAMRA-Phosphor-3′] (position 532–557). These primers were

tested in the GenBank database and

found to be pan-specific and to cover

all enteroviruses present in GenBank.

The reaction mixture (final volume

25 μL) was prepared in a single tube as

follows: 1× TaqMan buffer A, 5.5 mM

MgCl2

(both from PE Biosystems,

Courtaboeuf, France); 500 μM dNTPs

(Roche Molecular Biochemicals); 500

nM reverse primer Ev1, 400 nM primer

Ev2 (both from Genosys, Pampisford,

UK); 120 nM Ev-probe (Eurogentec,

Serraing, Belgium); 6% glycerol (Prolabo); 1.7% PVP-25 (Serva, Paris,

France); 1.5 μg T4 gene 32 protein

(Amersham Pharmacia Biotech, Orsay,

France); 5 UI MuLV and 2.5 UI AmpliTaq Gold (both from PE Biosystems)

and 10 UI RNasin (Promega, Charbonnière, France). Twenty microliters of the

reaction mixture were added to the PCR

tubes containing 5 μL RNA from one of

the sludge samples or RNA from the

constructed standard in serial dilution.

Enterovirus RNA was reverse transcribed into cDNA (45 min at 50°C) and

the 147-bp fragment was amplified by

PCR (45 cycles of 94°C for 15 s and

60°C for 1 min) on an ABI Prism 7700.

Enterovirus RNA Standard for

Absolute Quantification

An RNA standard representing the

5’NCR of enterovirus RNA was synthesized in vitro as follows: cDNA from

Vol. 29, No. 1 (2000) BioTechniques 89
Mahoneytype1polioviruswas amplified

byPCRusingprimerEv1Clon5′-TGGCCAATCGAATTCGCTTTA-3′(position

612–632) and primer Ev2Clon 5′-CTACATAAGGATCCTCCGGCC-3′(position 432–452) modified by the introduction of restriction sites (BamHI and

EcoRI). The 201-bp fragment was identified on a 1.5% agarose gel, excised,

isolated from the gel and ligated into the

polylinker of a pT7-blue vector (Novagen, Madison, WI, USA). The recombinant plasmid (pT7-EV) was transformed

into E. coli (Novagen) and a positive

colony was amplified in LB medium

containing tetracycline and ampicillin.

Plasmids were purified by alkaline lysis

(2), linearized, purified by gel electrophoresis and extracted from the gel

using a plasmid maxi kit (Qiagen).

Standard plasmids were quantified

using spectrophotometric analysis. The

sequence and orientation were verified

by automatic sequencing. Linearized

plasmid (1.5 μg) was used to perform

an in vitro transcription reaction with

T7 DNA polymerase (Roche Molecular

Biochemicals) at 37°C for 2 h. After digestion with RNase-free DNase, the resulting RNA transcripts were purified

with phenolchloroform and precipitated

with isopropanol. The RNA transcripts

were dissolved in sterile, RNase-free

water and quantified by spectrofluorometric analysis using Ribogreen (Interchim, Montluçon, France). The titer

was 1010 copies/μL. Dilutions (five

copies to 2 × 107) of the RNA standard

were made in TE buffer containing

RNasin. Aliquots of the diluted RNA

stock were frozen immediately at -

70°C. Each aliquot was used once only

for a fluorogenic RT-PCR.

Analysis of Fluorescence Signals on

the ABI Prism 7700

Real-time fluorescence measurements were taken and the threshold cycle (Ct

) value for each sample was calculated by determining the point at which

fluorescence exceeded a threshold limit

(10 times the baseline SD). A standard

graph of the Ct

values obtained from the

serially diluted external RNA standard

was compiled. The correlation coefficient and the slope were calculated. Ct

values from the sludge samples were

plotted on the standard curve, and the

number of copies was calculated automatically by Sequence Detector v1.6.

Samples were defined as negative if

their Ct

values exceeded 45 cycles.

RESULTS AND DISCUSSION

During the development of this

method, the optimal conditions for RTPCR were determined by amplifying a

range of the standard RNA. A problem

we encountered was that of sensitivity

(Figure 1A; detection limit = 1.6 × 104

copies) related to the secondary structures present in the 5′ NCR of the enterovirus genome. For this reason, we

decreased the RT/Taq ratio and used the

T4 gene 32 protein, usually considered

to be a ssDNA binding protein and a

dsDNA helicase (11,3). Figure 1B

demonstrates the benefits of adding this

protein to the reaction mixture (in quantities of 1.5 μg/reaction) on the amplification yield of the standard RNA.

The detection limit, intra-assay and

Short Technical Reports

90 BioTechniques Vol. 29, No. 1 (2000)

Figure 1. Comparison of intensity and amplification quality in sludge extracts. These extracts contains seven successive fivefold dilutions of standard RNA (25 to 4 × 10

5 copies) in the presence and absence of T4 gene 32 protein (Fam C3-D3 = 25, Fam C4-D4 = 125, Fam C5-D5 = 640, Fam C6-D6 = 3.2

× 10

3, Fam C7-D7 = 1.6 × 104, Fam C8-D8 = 8 × 104, Fam C9-D9 = 4 × 105). 1A: Amplification in the

absence of T4 gene 32 protein, three dilutions are amplified. 1B: Amplification in the presence of T4

gene 32 protein, all dilutions are amplified.

A

B
interassay variability were determined in

10 replicates of tenfold dilution standards, from 5 to 5 × 106 RNA copies,

amplified in eight independent assays.

Intra-assay CVs varied between 6.34%

and 0.74%, and interassay CVs between

4.51% and 0.66%. The values of 6.34%

and 4.51% obtained with low RNA concentrations agree with those found in

other TaqMan reactions (3.4% and

6.2%) (6,14). A variability of five copies

of the standard is acceptable, and no negative result was obtained. The sensitivity

of the RT-PCR is therefore five copies.

Other problems encountered were

those caused by the complexity of the

material, such as the residual sludge.

Sludges contain numerous inorganic

and organic compounds—humic acids

and polyphenols, for example—which

are liable to form complexes with nucleic acids and inhibit amplification enzymes. Figure 2 shows the presence of

such inhibitor substances on the graphs

fam-D1 produced by the Sequence Detector software when 8 × 104 copies of

enterovirus RNA from a sludge sample

were amplified. PVP is described in the

literature as forming complexes with

phenolic compounds (9,26). For maximum effect, it was added both at the

RNA extraction stage and to the PCR

mixture (Figure 2, Fam-D6). The

amount of PVP required in the RNA

extraction process was fixed at 2% as in

the cetyltrimethylammonium bromide

(CTAB) extraction protocol (22), while

the amount required in the amplification mixture was determined by adding

various quantities between 1% and 2%,

as did Koonjul et al. (9). The optimum

PVP amount was found to be 1.7%.

The analytical sensitivity of our

method, considering all extraction

steps, is 2000 copies/10 g (dry solids)

of sludge. However, lower numbers can

be found because the linearity of the

standard curve permits the interpolation

of values below the lower limit of the

dynamic range (14). Therefore, the theoretical detection limit of our RT-PCR

can be adjusted to 400 copies/10 g (dry

solids). This sensitivity is compatible

with requirements accepted in the literVol. 29, No. 1 (2000) BioTechniques 91

Results from RT-PCR Results from cell culture

Samples (number of copies/10 g dry solids) (MPNCU/10 g dry solids)

control 1 (7 × 104 pfu) 1.5 × 106 4.9 × 104

control 2 (7 × 104 pfu) 1.4 × 106 3.5 × 104

control 3 (7 × 104 pfu) 8 × 106 3.4 × 104

control 4 (7 × 104 pfu) 1 × 106 1.4 × 104

control 5 (7 × 104 pfu) 7 × 106 2.2 × 104

control 6 (7 × 103 pfu) 2 × 105 1.6 × 103

control 7 (7 × 103 pfu) 3 × 105 3.6 × 103

control 8 (7 × 103 pfu) 1 × 105 1.7 × 103

1P 54 720 10

2P 230 400 2242

3P 115 200 326

4A 5664 107

5A 14 400 278

6A <400 < 3

7T 14 400 < 3

8T 14 400 72

9T < 400 < 3

Measurements use RT-PCR and cell culture in eight sludge samples artificially

contaminated with 7 × 104 and 7 × 103 pfu poliovirus 1 and nine natural sludge

samples of three different types (P = primary, A = activated, T= thickened).

Table 1: Enterovirus Quantification Measurements.
ature as suitable for the detection of enteroviruses in sludge from waste-water

treatment plants (10,20,23,24) .

This fluorogenic RT-PCR protocol

was first evaluated on eight samples of

artificially contaminated thickened

sludge and then applied to nine natural

sludge samples. The results, presented

in Table 1, were compared to measurements made of virus quantities by

means of cell culture. For the samples

artificially contaminated with 7 × 104

or 7 × 103 pfu, the mean values as measured by RT-PCR and cell culture, respectively were 3.78 × 106 copies/10 g

and 3.08 × 104 MPNCU/10 g (n = 5) or

2 × 105 copies/10 g and 2.96 × 103 MPNCU/10 g (n = 3) (Table 1). In the natural sludge samples, two samples were

negative (< 400 copies/10 g) according

to RT-PCR and three according to cell

culture (< 3 MPNCU).

The quantity of genomic RNA measured was always larger than the number of infectious particles, being on average 150 times greater for natural

sludge and 120 times greater for contaminated sludge. This can be explained

by (i) the aggregation of infectious particles into clumps that, while consisting

of several viruses, only cause one area

of lysis in a cell culture; (ii) the variable

effects of cell culture inhibitory substances that might still be present in the

concentrates; and (iii) the ability of RTPCR to detect inactivated viruses. These

results, which agree with those reported

in the literature (8,18,19), show a sharp

decrease in quantities of virus present

from primary sludge through activated

and thickened sludges.

The fluorogenic RT-PCR technique

developed in this study allows real-time

analysis of the amplification reaction

and calculation of genomic RNA quantities at the start of the exponential phase

of amplification. The fluorescence signal caused by cleavage of the TaqMan

fluorogenic probe can only be generated

if the target sequence has been amplified, which gives the reaction a high level of specificity. Our protocol is sensitive, reproducible and rapidly executed

on multiple samples. These features

make it an excellent large-scale screening tool for sludge from waste-water

treatment plants and for monitoring

sewage processing procedures, even

though the presence of infectious particles must be confirmed by cell culture,

as current regulations require.

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Figure 2. Graph showing fluorescence intensity as a function of numbers of amplification cycles. Comparison of amplification intensity of the same sludge concentrate (10 μL) containing 8 × 10

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This study is based on work supported by

A.d.e.m.e (the French environment and energy management agency) and Anjou Recherche. We thank INSERM U437 and Perkin

Elmer for technical support with the ABI

Prism 7700. Address correspondence to Dr.

Serge Monpoeho, Laboratoire de Virologie

CHU Hotel Dieu, 9 quai moncousu, 44035

Nantes, France. e-mail: sbillaudel@chunantes.fr

Received 23 July 1999; accepted 15

March 2000.

S. Monpoeho, A. Dehée

2

, B.

Mignotte, L. Schwartzbrod

1

,

V. Marechal

2

, J.-C. Nicolas

2

, 

S. Billaudel and V. Férré

Laboratoire de Virologie CHU

de Nantes, Nantes

1Laboratoire de Virologie

faculte de pharmacie

Nancy, Nancy

2Laboratoire de Virologie

hopital Rothschild

Paris, France