Rapid and sensitive detection of peste des petits ruminants virus by a polymerase chain reaction assay

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Abstract

A rapid and specific test was developed for the diagnosis of peste des petits ruminants disease. This assay is based on the rapid purification of RNA on glass beads followed by the reverse transcription-polymerase chain reaction (RT-PCR). To that effect, a set of primers (NP3/NP4) was used to amplify specifically a fragment of about 350 bp in the 3′ end of the RNA messenger that encodes the nucleocapsid protein of the peste des petits ruminants virus. The PCR-product was detected by UV illumination after electrophoresis on agarose gel or by hybridisation with a digoxigenin-11-dUTP labelled oligonucleotide probe after a blot transfer. In comparison with the conventional titration technique on Vero cells, this RT-PCR assay was 1000-fold more sensitive. Compared with the popular Chomczynski and Sacchi's method [Anal. Biochem. 162 (1987) 156], the purification of the RNA on the glass beads offers the advantage of being more rapid and also avoiding the use of solvents.

Introduction

Peste des petits ruminants (PPR) is a severe disease of goats and sheep with high morbidity and sometimes high mortality. It continues to cause serious economic losses in these species in Africa, the Middle East, and Asia (Taylor, 1984, Shaila et al., 1989, Lefevre and Diallo, 1990, Abu-Elzein et al., 1990, Nanda et al., 1996, Govindarajan et al., 1997). The causative agent is a morbillivirus, peste des petits ruminants virus (PPRV). It is closely related to rinderpest virus (RPV), another member of the morbillivirus genus which can also cause a disease in small ruminants (Anderson et al., 1990, Couacy-Hymann et al., 1995). At present, rinderpest eradication projects are carried out in Africa, the Middle East and Asia. However, for the diagnosis of rinderpest in small ruminants, it is essential to differentiate it clearly from PPR. This is impossible clinically because the symptoms of both diseases are very similar. In the laboratory, this is possible by virus isolation followed by either the differential neutralisation test in cell culture or by the animal inoculation because PPRV causes inapparent infection in cattle whereas small ruminants can show clinical symptoms after RPV infection. These techniques are all time-consuming. Moreover, RPV infection of small ruminants is sometimes asymptomatic (Couacy-Hymann et al., 1995). In the last decade, molecular biology techniques have permitted the development of specific and sensitive tests for rapid diagnosis. The structure and biology of peste des petits ruminants is now well known. As with other Morbilliviruses, it has an unsegmented negative stranded RNA genome encoding six structural and two non-structural proteins (Haffar et al., 1999). Among them, the nucleocapsid protein (NP) is the major viral protein. It has been the target for developing diagnostic tests that can be used to distinguish rinderpest infection from PPR. Monoclonal antibodies, anti-NP, were used to develop ELISAs for both specific serological diagnosis in the competitive format and for antigen detection by immunocapture (Libeau et al., 1994, Libeau et al., 1995). For this latter test, the limit of detection has been estimated at 100.6 TCID50 of virus in 50 μl of sample. In 1989, nucleic acid technology was applied for the first time to the detection of RPV and PPRV by using, as probes, the cDNA corresponding to the nucleocapsid gene of each virus and labelled with 32P nucleotide (Diallo et al., 1989). Although sensitive, such a test is not suitable for routine use in most of the developing countries due the health hazard linked to the radioactive labels and the lack of adequate equipment. An alternative and very sensitive technique, the amplification of the viral nucleic acid by the reverse transcription-polymerase chain reaction (RT-PCR) is described for specific detection of PPRV. In this test, we use a simplified RNA extraction technique instead of the classical phenol–chloroform extraction method.

Section snippets

Virus

Nineteen PPRV isolates available in CIRAD (Montpellier, France) were grown in Vero cells: Guinea Bissau, Senegal (94), Burkina-Faso (86), Côte-d'Ivoire (89), Guinea, Dorcas (Sultanate of Oman), Ibri (Sultanate of Oman), Sinnar (Sudan), Ethiopia 94, Nigeria 75/1, Nigeria 75/2, Nigeria 75/3, Nigeria 76/1, Accra, Egypt, Ghana 78/1, Israel, Central Africa Republic, India 94/1, India Calcutta, India Pradesh. Rinderpest attenuated vaccine strain has also been used. Cell growth medium was Eagle's MEM

Rapid extraction of RNA

Attempts were made to simplify the RNA extraction method, a step critical for RT-PCR technique. The glassmilk was used as RNA binding matrix in different conditions: guanidine thiocyanate or sodium iodide solutions as lysis buffers and RNase inhibitors, different times of incubation as indicated in materials and methods. The RNAs that were extracted under different conditions from PPRV infected cells then dot-blotted and revealed by PPRV NP radioactive probe. The results obtained from this

Discussion

PPRV and RPV infections of small ruminants are clinically similar so the differential diagnosis has to be made between these two diseases when they coexist in an area. Such a diagnosis could be made now by competitive ELISA (antibody detection), or by immunocapture (antigen detection). Shaila et al. (1996) have reported the application of the RT-PCR to detect PPR RNA in pathological samples. To purify the RNA, they used the phenol–chloroform method described by Chomczynski and Sacchi (1987)

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