Establishment and application of a point-of-care testing and diagnosis method for early immediate expression gene IE1 of cytomegalovirus in maternal urine based on isothermal amplification

Abstract

Background: Human Cytomegalovirus virus (HCMV) is a worldwide virus that causes no serious symptoms in most adults. However, HCMV infection during pregnancy, it may lead to a series of serious complications, such as hearing loss, mental retardation, visual impairment, microcephaly and developmental retardation.

Aim: The aim of this study was to develop a simple, low dependence on equipment and accurate method for HCMV detection based on the recombinase polymerase amplification (RPA) and lateral flow chromatography strip (LFS) reading.

Methods: In order to meet the feasibility of HCMV early screening, three pairs of RPA primers were designed based on the UL123 gene encoding IE1, which was expressed immediately in the early stage of HCMV. In order to improve the specificity of the reaction and satisfy the visual detection, a specific probe was designed to insert THF site between upstream and downstream primers, fluorescein isothiocyanate (FITC) and C3spacer were used to modify the 5' end and the 3' end respectively, and Biotin was used to modify the 5' end of the reverse primer. HCMV standard strain AD169 was enriched by ARPE-19 cells culture, and its genome was extracted. The primers and probes were screened by RPA-LFS test, and the optimal reaction temperature and time were determined The specificity was verified in different viruses, bacteria and parasites. The standard curve was drawn based on the constructed recombinant plasmid of pMD18T-HCMV-UL123 and used for HCMV genomic DNA quantification and determination of the detection sensitivity. Urine samples from artificial HCMV contamination or clinical collection were prepared to evaluate the consistency with the results of real-time quantitative PCR.

Results: The results showed that the primers and probes for HCMV RPA-LFS detection based on UL123 gene were successfully screened, the amplification of HCMV genomic DNA with as low as 30 copies could be completed at 37 °C within 15 min, it did not react with Human herpesvirus 1, Streptococcus pyogenes, Candida albicans, Listeria monocytogenes, Y. enterocolitica, Klebsiella Pneumoniae, Enterobacter cloacae, Citrobacter freundii, Vibrio alginnolyfificus, Vibrio parahaemolyticus, S. typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa and Trichomonas vaginalis. The positive rate of PCR was 96.67 % in 30 simulated urine samples and 100 % in 127 clinical urine samples with the same UL123 gene detection.

Conclusions: To sum up, we developed a diagnostic method for HCMV based on UL123 gene combined with RPA and LFS, which is low dependent on equipment, fast, sensitive and specific, provide reference for point-of-care testing HCMV in grass-roots laboratories and remote areas.

Keywords: Early immediate expression gene; Human cytomegalovirus; Isothermal amplification; Lateral flow chromatography strip; THF.

Fig. 1

Performance of primers and probe sets for HCMV amplification by RPA-LFS. HCMV-UL123-F1/R1/P, HCMV-UL123-F2/R2/P, and HCMV-UL123-F3/R3/P were used to amplify HCMV genomic DNA and detect LFS. The primers and probe names were labeled on the LFS. The C marked on the LFS side stands for control line, and the T stands for test line. NTC represents a negative control without adding a template.

Fig. 2

Screening of RPA amplification conditions based on HCMV-UL123-F3/R3/P primer probe pairs. Apply HCMV-UL123-F3/R3/P primer probe pairs within the temperature range of (A) 15, 20, 25, 30, 37, 39, 42, 45 °C and (B) 5, 10, 15, 20, 25, 30, 35, and 40 min, respectively. NTC represents a negative control without adding a template.

Fig. 3

The results of interspecies specificity of HCMV RPA-LFS. Purified HCMV, HSV, S. pyogenes, T. vaginalis, C. albicans, L. monocytogenes, Y. enterocolitica, K. Pneumoniae, E. cloacae, C. freundii, V. alginnolyfificus, V. parahaemolyticus, S. typhimurium, S. aureus and P. aeruginosa genomic DNA were used as templates, and negative control NTC without template was set. HCMV RPA-LFS was used to amplify HCMV at 37°C for 25 min, and then detected by LFS. Each template is labeled at the location of the corresponding strip.

Fig. 4

Minimum detection limit results of HCMV RPA-LFS detection method. (A) Establish a standard curve based on the pMD18T-HCMV-UL123 standard. Using pMD18T-HCMV-UL123 plasmids ranging from 3 × 10⁸ copies/mL to 3 × 10² copies/mL as templates, Ct values were determined by qPCR and standard curves were plotted. (B) Determination of the minimum detection limit for the HCMV RPA-LFS detection method. Using HCMV genomic DNA from 3 × 10⁴ copies/µL to 3 × 10⁻¹ copies/µL as templates, RPA-LFS detection was performed. The concentrations of each template are labeled above each experiment, with NTC as the negative control.

Fig. 5

Consistency evaluation of HCMV RPA-LFS detection method and qPCR method in spiked urine samples (A) HCMV RPA-LFS test results. (B) Heat map of qPCR test results.