A real-time PCR assay for bat SARS-like coronavirus detection and its application to Italian greater horseshoe bat faecal sample surveys

Abstract

Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population.

Figure 1

Distribution of bat sampling in Italy. Sites of bat sampling from August to November 2009. For correspondence between letters and places, see Table 1.

Figure 2

Multiple sequence alignment of primer binding sites. The alignments include 10 SARS-related coronavirus reference strains: SBRp3 (Bat SARS CoV Rp3/2004, DQ071615, identified from Rhinolophus pearsoni), SBRs (SARS coronavirus Rs_672/2006, FJ588686, identified from Rhinolophus sinicus), SHTor2 (SARS coronavirus Tor2, AY274119, identified from Human), SC (Civet SARS CoV SZ16/2003, AY304488, identified from Paguma larvata), SB (SARS coronavirus isolate CFB/SZ/94/03, AY545919, identified from Melogale moschata), SBRf1 (Bat SARS CoV Rf1/2004, DQ412042, identified from Rhinolophus ferrumequinum), SBRm1 (Bat SARS CoV Rm1/2004, DQ412043, identified from Rhinolophus macrotis), SBHKU3-1 (Bat coronavirus HKU3, DQ022305, identified from Rhinolophus sinicus), SB273 (Bat CoV 273/2005, DQ648856, identified from Rhinolophus ferrumequinum), and SB279 (Bat CoV 279/2005, DQ648857, identified from Rhinolophus macrotis).

Figure 3

Real-time PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: serial 10-fold dilutions of recombinant plasmid. Specific bands of approximately 168 bp were visualised for all replicates of recombinant plasmid dilutions, except the concentration of 1 × 10⁻¹ copies/μL. For recombinant plasmid dilution with a concentration of 1 × 10⁻¹ copies/μL only one specific amplicon to three replicates was visualised. MK: MassRuler Low-Range DNA Ladder (Fermentas, Burlington, Ontario, Canada). a, b, c, d, and e: recombinant plasmid dilutions with concentrations of 1 × 10³, 1 × 10², 1 × 10¹, 1 × 10⁰, and 1 × 10⁻¹ copies/μL, respectively. W: no template control (water).

Figure 4

Melting curve analysis of standard plasmid dilutions and sample 893/09-11. In gray: signals obtained from the standard plasmid dilutions; in black: signals obtained from the sample 893.09-11; derivative −dF/dT where F is fluorescence and T is time; °C: temperature (centigrade).

Figure 5

Real time-PCR reaction products checked on agarose gel stained with ethidium bromide in standard tris-acetate-EDTA (TAE) buffer: positive samples detected by developed real-time PCR. Specific bands of approximately 168 bp were visualised for all replicates of the detected positive samples. In this figure is represented the 2% (w/v) agarose gel electrophoresis of five of the 11 positive samples detected in bats belonging to sampling area A (San Cesario sul Panaro, MO). Pl: amplicon of the recombinant plasmid with 1 × 10⁴ copies/μL; 1, 2, 3, 4, and 5: amplicons of the five positive samples belonging to sampling area A, each with three repetitions.