A ONE -TUBE NUCLEIC ACID EXTRACTION AND AMPLIFICATION (ERTPCR) METHOD FOR DETECTING RNA

VIRUSES

NIGEL ~00~1.~3, ANDREW s. KURDZIEL~J, JOHN P. HAYS.^, SUSAN H. GORDON', BRYAN J. DONALD' and STEVEN H. MYINF

Virology and Molecular Methods, Food Microbiology Group, Central Science Laboratory, Ministry of Agriculture, Fisheries and Food.

Sand Hutton, York YO41 lLZ, UK

9epartment of Microbiologv and Immunology, University ofkicester. Leicester LEI 4HN, UK

Accepted for Publication February 23,1999

ABSTRACT

Extraction Reverse Transcription Polymerase Chain Reaction (ERTPCR), a rapid and simple method which allows detection of RNA viruses, is described. Unlike conventional Reverse Transcription Polymerase Chain Reaction(RTPCR), which requires extraction of viral nucleic acid prior to reverse transcription and cDNA amplification, ERTPCR allows these procedures to be accomplished in one step in the same reaction tube. This is facilitated through heat extraction of viral RNA, and the use of a thennostable enzyme which can perjbrm reverse transcription and cDNA amplification in the same bufler system. The technique is at least as sensitive as a standard RTPCR. Its use in detecting rotavirus in feces, and hepatitis A virus in infected tissue cultures, is demonstrated. A silica spin column was used to remove RTPCR inhibitors fiom these samples.

INTRODUCTION

Nucleic acid amplification has now become established as part of the range of techniques available to scientists for the detection of viruses in clinical and environmental samples (Newton and Graham 1994; Cook and Myint 1995; Myint 1995). Generally, nucleic acid is prepared by chemical extraction of the sample before applying the amplification techniques (Le Guyader 1994; Easton et al. 1994). Applying extraction procedures necessary to obtain viral nucleic acid from

'All correspondence to be addressed to this author.

Journal of Rapid Methods and Automation in Microbiology 7 (1999) 61-68. All Righrs Reserved. *Copyright 1999 by Food & NulriZion Press. Inc., Trumbull, Connecficuf. 61

62 N. COOK ETAL.

samples inevitably leads to some loss of target, which reduces the sensitivity of &kction; and potential canyover of extraction chemicals, e.g. phenol, may affect subsequent enzymatic reactions.

Once RNA is liberated from the viral capsid it can be susceptible to degradation by FWAse enzymes, and special treatment of reagents and laboratory ware is required to control this (Farrelll993). RNA viruses require reverse transcription (RT) pnor to PCR, and this has normally been achieved by the use of a separate enzymatic procedure using heat labile retrovirus - derived enzymes (Le Guyader 1994; Jonassen et al. 1995). This necessitates opening the reaction tube each time a separate enzyme is required, but the more a sample is thus handled, the more potential it has for acquiring contamination from other sources. It would be advantageous to have simpler procedures for viral detection, especially where routine analysis is required. The use of chemicals can in fact be avoided through liberation of RNA by heat denaturation of the viral capsid (Jonassen et al. 1995), and thermostable DNA polymerases with reverse transcriptase activity have become available (Meyers and Gelfand 1991; Easton et al. 1994). A one - tube extraction, reverse transcription and amplification method has been used which employs antigen - capture (Jansen et al. 1990; Deng et al. 1994). However, after heat denaturation' of the captured viral capsids, reagents for the subsequent reactions must be added in separate steps.

The method presented here allows all the steps from extraction of viral RNA to amplification to be performed in a single reaction tube, without having to open it after the addition of the virus and the necessary reagents. To facilitate this, a system is employed in which a thermostable enzyme can perform both reverse transcriptase and DNA polymerase activities in the same buffer. The sample containing the virus particles is mixed with a concentrated solution of the reaction reagents, and the tube is sealed. Then, the sample is heated to disrupt the viral capsids, the temperature subsequently lowered to permit reverse transcription, and finally thermocycling allows amplification of the transcribed DNA.

The following report demonstrates that the ERTPCR method is comparable in sensitivity to the standard, uncoupled RTPCR approach. As with other amplification reactions, it can be susceptible to inhibition by substances present in clinical and environmental samples; however, the report details a system by which this may be combated.

MATERIALS AND METHODS

Preparation of Poliovirus Suspension

Poliovirus l a was kindly provided by Professor C.R. Madeley and Mr. F. Laidler of Newcastle - upon - Tyne PHLS. The viruses were propagated on MA 104

DETECTION OF RNA VIRUSES 63

cells (a transformed monkey kidney cell line) until visible CPE, whereupon they were concentrated by PEG - precipitation and ultracentrifugation of 21 infected culture. The suspension was quantified by electron microscopy, using a suspension of 80 nm latex beads (Agar Scientific Ltd.) as a standard.

Standard RT PCR

Serial 10-fold dilutions of the poliovirus suspension were made in sterile distilled water. 10 pL of each dilution was added to 10 pL of RT mix (containing 4 pL Of 5 x AMV buffer [Promega], 1 pM downstream primer Po2 (Egger er al. 1996) 15 U RNA Guard [Pharmacia Biotech], 200 pM each dNTP, and 2.5 mM dithiothreitol). The suspension was incubated at 94C for 5 min to disrupt the viral capsids, then immediately placed on ice. After 5 min, 1 U avian myeloblastosis reverse transcriptase (AMV, Promega) was added, and the reaction incubated at 95C for 5 min, then at 42C for 1 h. The contents of the reaction were then added to 30 pL of PCR mix (10 x PCR buffer [Promega], 2 mM MgCl,, 200 pLM each dNTP, 0.25 pM Pol and Po2 (Egger et ul. 1996) and incubated at 94C for 5 min. 2.5 U Tuq polymerase (Promega) was then added, and the reaction themocycled as follows: 94C 1 min, then 50 cycles of 94C 45 s and 60C 45 s, followed by a final extension step of 72C for 7 min. The product was visualized by electrophoresis of 10 pL of the reaction mix on a 2% agarose gel containing 5 ng mL-' ethidium bromide, and UV transillumination. The expected product size is 193 bp.

ERTPCR

Ten pL of each poliovirus suspension dilution was added to 15 pL H20 in a sterile reaction tube. Then, 25 pL of a mixture containing 2 x EZ buffer (Perkin Elmer), 400 pM each dNlT, 0.5 pM primers Pol and P02,1.25 U rTth polymerase (Pakin Elmer), and 4 mM Mn(OAc), was added. The mixture was thermocycled as follows: 94C for 5 min (capsid disruption), 60C 1 h (reverse transcription), 94C 1 min (initial cDNA denaturation), then 50 cycles of 94C 45 s and 60C 45 s to amplify the cDNA, followed by a final extension step of 72C for 7 min. The product was visualised as described above.

Detection of Rotavirus in Feces

A fecal sample from a child with gastroenteritis was obtained from Leicester Public Health Laboratory Service. 100 mg was suspended in 1 mL pH 7.0 buffer (Sigma), vortexed 30 s, and spun at 13,000 x g for 10 min in a microfuge to remove particulates. 25 pL of the supernatant was used in an ERTPCR reaction. The rest of the supernatant was added to 4 mL pH 7.0 buffer, then passed, by synnge, through a mini spin column (Wizard, Promega) containing silica (NP40, Amicon). The column was spun to dryness at 13,000 x g for 10 s, then 100 pL 1

64 N. COOK ETA&

x EX buffer was added to it. After 2 min at ambient temperature, the column was spun at 13,000 x g for 30 s, and the eluate collected. 25 pL was then used in an ERTPCR reaction. Each reaction contained 1 x EZ buffer, 1.5 mM Mn(OAc),, 200 pM each dNTp, 1.25 U rTrh, and 0.5 pM primers H1 and H2 (LeGuyader ef al., 1994). The temperature regime was 1 min at 86C, 1 h at 55C, 1 min at 94C, followed by 35 cycles of 94C for 45 s and 57.5C for 45 s. A final extension at 72C for 7 min was followed by a soak at 4C The product was visualized as described above. The expected product size is 247 bp.

Detection of Hepatitis A Virus In Infected Cell Culture Supernatant

Hepatitis A virus Hh4175 was obtained from the American Type Culture Collection. It was supplied as a suspension in cell culture medium. 10 pL of this suspension was added to 5 mL pH 7.0 buffer, then passed through the silica min spin column as described above. The virus particles were eluted from the column with 100 pL 1 x EZ buffer, and 25 pL eluate was used in an ERTCR reaction. As a control, to demonstrate the effectiveness of the column at removing inhibitors, 10 pL of the original virus suspension was added to 90 pL 1 x EZ buffer, and 25 mL of this was used in an ERTPCR reaction. Each reaction contained 1 x EZ buffer, 2 mM Mn(OAc),, 100 pM each dNTP, 1.25 U rRh, and 0.5 pM primers H1 and H2 (LeGuyader et al. 1994). The temperature regime was 1 min at 94C, 45 min at 60C, 1 min at 94C, followed by 40 cycles of 94C for 45 sec and 55C for 45 sec. A final extension at 72C for 7 min was followed by a soak at 4C. The product was visualized as described above. The expected product size is 392 bp.

RESULTS

Figure 1 shows a comparison of the results obtained by applying standard RTPCR and ERTPCR to dilutions of a poliovirus suspension. The reactions shown contained 9000,900,90,9 and 4 virus particle each. Both methods produced clear signals from the reactions containing 90 virus particles, and a faint signal from the ERTPCR containing 9 particles may be seen. Thus, the detection sensitivity achieved by ERTPCR is at least as high as that of the standard method.

Figure 2 shows that viruses may be detected by ERTPCR in samples which contain inhibitory substances, when the reaction is preceded by spin column purification. This was demonstrated by assaying a fecal sample forthe presence of rotavirus. When untreated fecal extract was used, no signal was obtained, but after passing the extract through the silica column a signal could be visualized. Similarly, hepatitis A virus detection in infected cell culture supernatant was hampered, probably by components of the growth medium. When these were removed by passage through the spin column. the viruses could be detected by ERTPCR.

DETECTION OF RNA VIRUSES 65

FIG. 1 , COMPARISON OF DETECTION SENSITIVITIES OF STANDARD RTPCR AND ERTPCR, USING POLIOVIRUS PARTICLES

A: Standard RTPCR. B: ERTPCR. The signals were obtained from reactions containing varying numbers of virus particles. Lane I , 100 bp ladder. Lane 2,9000 virus particles. Lane 3,900 virus particles. Lane 4.90 virus particles. Lane 5.9 virus particles. Lane 6. < 1 virus particle. Lane 7,

negative control (H20).

DISCUSSION

The ERTPCR method is easy to perform, requiring no chemical extraction, it is rapid, taking only a few hours from sample addition to detection, and it has several advantages over current protocols. As it is not necessary to open the tube

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FIG. 2. DETECTION OF ROTAVIRUS IN FECES, AND HEPATITIS A VIRUS IN INFECTED CELL CULTURE SUPERNATANT, BY ERTPCR

Lane I , 100 bp ladder. Lane 2, rotaviruscontaining fecal extract, no column purification. Lane 3, rotaviruscontaining fecal extract, with column purification. Lane 4, hepatitis A virus-infected cell

culture supernatant, no column purification. Lane 5. hepatitis A virus-infected cell culture supernatant, with column purification.

between addition of the initial components and removal of the amplified target for detection, the risk of cross contamination between samples is minimized. Also, as RNA is liberated from the protective viral capsid at high temperature, and subsequent reverse transcription is performed above 60C, the risk of degradation by RNases which may be present in the sample is removed, and the use of hannful

DETECTION OF RNA VIRUSES 67

chemicals, such as diethyl pyrocarbonate, obviated.. A method to perform heat extraction and RTPCR was described by Wang et al. 1996, in which mouse hepatitis virus was used as the target in a reaction carried out in an air thermocycler. However, capsid disruption was performed at 50C, and it is unlikely that more robust viruses would be detected by that method, since the retroviral reverse transcriptases it employed would be inactivated at the temperatures required for capsid disruption.

The removal of inhibitory substances from the samples by using the spin column is mediated by adsorptiodelution of the virus particles to the silica. At neutral pH, enteric viruses have a net negative charge, and will bind to the positively charged silica particles (Gerba 1984), while inhibitory substances are passed through. Virus elution is achieved by raising the pH, and the EZ buffer, at pH 8.5, is suitable for this. Silica spin column purification is thus a rapid and convenient procedure for clean-up of samples prior to virus detection by ERTPCR.

It is anticipated that ERTPCR will work with many types of samples, both clinical and environmental. It is employed routinely in our laboratories, where rapid methods for detecting viruses in foodstuffs and aquatic systems are under development. It should also be useful where cell culture is used to propagate viruses from difficult clinical or environmental samples prior to detection and identification by RTPCR (Egger et al. 1995). The silica column purification method is a useful way of immediate removal of inhibitory substances; however, if desired, ERTPCR should, be compatible with initial antigen capture of the virus as an alternative. Finally, the simplicity of the method may lend itself to automation, facilitating routine virus screening in high throughput laboratory systems.

ACKNOWLEDGMENTS

NC, ASK, JPH, SHG and BD were supported by a grant from the Ministry of Agriculture, Fisheries and Food. At the University of Leicester, NC, ASK and JPH were supported by grants from the Science and Engineering Research Council, the Department of Trade and Industry, and the Medical Research Council, respectively.

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