Development and evaluation of recombinase-aided amplification assays incorporating competitive internal controls for detection of human adenovirus serotypes 3 and 7

Abstract

Background: Human adenoviruses are a common group of viruses that cause acute infectious diseases. Human adenovirus (HAdV) 3 and HAdV 7 cause major outbreaks of severe pneumonia. A reliable and practical method for HAdV typing in clinical laboratories is lacking. A simple, rapid and accurate molecular typing method for HAdV may facilitate clinical diagnosis and epidemiological control.

Methods: We developed and evaluated duplex real-time recombinase-aided amplification (RAA) assays incorporating competitive internal controls for detection of HAdV 3 and HAdV 7, respectively. The assays were performed in a one-step in a single tube reaction at 39° for 20 min.

Results: The analytical sensitivities of the duplex RAA assays for HAdV 3 and HAdV 7 were 5.0 and 14.8 copies per reaction, respectively (at 95% probability by probit regression analysis). No cross-reaction was observed with other types of HAdV or other common respiratory viruses. The duplex RAA assays were used to detect 152 previously-defined HAdV-positive samples. These results agreed with those obtained using a published triplex quantitative real-time PCR protocol.

Conclusions: We provide the first report of internally-controlled duplex RAA assays for the detection of HAdV 3 and HAdV 7. These assays effectively reduce the rate of false negative results and may be valuable for detection of HAdV 3 and HAdV 7 in clinical laboratories, especially in resource-poor settings.

Keywords: Detection; Duplex recombinase aided amplification; Human adenovirus; Pneumonia.

Fig. 1

a Amplification curves 1 to 3 indicate a dilution range from 10¹ to 10³ copies/reaction of the internal control (IC) with 100 copies of HAdV 3 standard plasmid. b Amplification curves 1 to 3 indicate a dilution range from 10¹ to 10³ copies/reaction of the IC with 100 copies of HAdV 7 standard plasmid

Fig. 2

A1: Amplification curves 1 to 5 indicate a dilution range from 10⁴ to 10⁰ copies/reaction of HAdV 3 standard plasmid with 1000 copies of the IC in the HEX (5-hexachlorofuorescein) detection channel. A2: Amplification curves for 1000 copies of the IC combined with (curves 1 to 5) 10⁴ to 10⁰ copies/reaction of HAdV 3 standard plasmid, in the FAM (6-carboxyfuorescein) detection channel. The same color amplification curves in A1 and A2 indicate use of the same pair of primers in the reaction tube. B1: Amplification curves 1 to 4 indicate a dilution range from 10⁴ to 10 copies/reaction of HAdV 7 standard plasmid with 1000 copies of the IC in the HEX detection channel. B2: Amplification curves for 1000 copies of the IC combined with (curves 1 to 4) 10⁴ to 10 copies/reaction of HAdV 7 standard plasmid, in the FAM detection channel. The same color amplification curves in B1 and B2 indicate use of the same pair of primers in the reaction tube

Fig. 3

A1 Amplification curves 1 to 4 indicate HAdV 3 clinical samples with triplex quantitative real-time PCR (tq-PCR) cycle threshold (CT) values of 16.48, 20.39, 36.42, and 36.45 with 1000 copies of the IC in the HEX detection channel. A2 Amplification curves for 1000 copies of the IC combined with (curves 1 to 4) samples as in A1, in the FAM detection channel. The same color amplification curves in A1 and A2 indicate use of the same pair of primers in the reaction tube. B1 Amplification curves 1 to 4 indicate HAdV 7 clinical samples with tq-PCR CT values of 15.85, 17.65, 33.57, and 35.99 with 1000 copies of the IC in the HEX detection channel. B2 Amplification curves for 1000 copies of the IC combined with (curves 1 to 4) samples as in B1, in the FAM detection channel. The same color amplification curves in B1 and B2 indicate use of the same pair of primers in the reaction tube

Fig. 4

Schematic diagram of duplex recombinase-aided amplification (RAA) assays for detection of HAdV 3 and HAdV 7