key: cord-0858449-16nfwoqt authors: Lai, Meng Yee; Bukhari, Fatma Diyana Mohd; Zulkefli, Nur Zulaikha; Ismail, Ilyiana; Mustapa, Nur Izati; Soh, Tuan Suhaila Tuan; Hassan, Afifah Haji; Peariasamy, Kalaiarasu M; Lee, Yee Leng; Suppiah, Jeyanthi; Thayan, Ravindran; Lau, Yee Ling title: Colorimetric detection of SARS-CoV-2 by uracil-DNA glycosylase (UDG) reverse transcription loop-mediated isothermal amplification (RT-LAMP) date: 2022-04-25 journal: Int J Infect Dis DOI: 10.1016/j.ijid.2022.04.036 sha: 87755ad48edef86daddfee1d53715884fdf2acd8 doc_id: 858449 cord_uid: 16nfwoqt Preventing RT-LAMP carryover contamination could be solved by adding deoxyuridine triphosphate (dUTP) and uracil- DNA glycosylase (UDG) into the reaction master mix. RNA was extracted from nasopharyngeal swab samples by a simple RNA extraction method. Out of 77 samples tested, we managed to obtain 91.2% sensitivity (95% CI: 78-98.2%) and 100% specificity (95% CI: 92-100%) using UDG RT-LAMP. This colorimetric UDG RT-LAMP is a simple-to-use, fast, and easy-to-interpret method. It could serve as an alternative diagnosis of SARS-CoV-2 infection, especially in remote hospitals and laboratories with under-equipped medical facilities. It has been more than two year since the new coronavirus (called severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) pandemic hit the world. Rapid diagnosis method such as reverse transcription loop mediated (RT-LAMP) (Notomi et al., 2000) is critically important to minimize the spread of the illness as well as protect the public. Due to LAMP's high sensitivity criteria, it is prone to carryover contamination. Fortunately, risks of carryover contamination can be reduced by incorporating deoxyuridine triphosphate (dUTP) and thermolabile uracil-DNA glycosylase (UDG) into the master mix (Hsieh et al. 2014) . By incorporating dUTP in the LAMP reactions, the amplified products will be chemically modified from the target DNA and specifically tagged with uracil. The subsequent LAMP reactions are treated with UDG, which makes only the natural target DNA intact for amplification and thus decreasing the risk of carryover contamination (Tang et al., 2016) . In this study, we included dUTP in RT-LAMP assay and compared its performance with dNTP RT- A total of 37 rRT-PCR positive (Ct value ranged from 15.51-38.85) and 40 rRT-PCR negative clinical swab samples were provided by Hospital Sungai Buloh and Institute for Medical Research, Malaysia. RNA was extracted using a Chelex 100 Resin concentration method as described by Janíková et al. (2021) and Perez et al. (2021) with some modification. The 30% (w/v) Chelex 100 Resin (Biorad Laboratories, USA) was prepared in TE buffer and vortex vigorously for 10 seconds and kept in 4°C until further use. Fifteen µL of samples was mixed with 22.5 µL of 30% (w/v) Chelex-TE. The tube was incubated in 98°C for 2 minutes followed by incubation on ice for 2 minutes. The sample was subjected to short spin and the supernatants were further mixed with 1.5 M sodium acetate and 0.3 M sodium 5 hydroxide (NaOH), pH 5.2. Three volumes of ice-cold ethanol were added, vortex for 5 seconds before being centrifuge for 10 minutes. The tube was air dried at room temperature for 10 minutes. The RNA pellet was re-suspended with 5 µL of TE buffer and the tube was vortex for 30 seconds before a quick spin. UDG RT-LAMP assay was performed in a total of 12.5 μL reaction mixture, Meanwhile, the dNTP LAMP mixture consisted of similar ingredients as mentioned in UDG RT-LAMP assay except for 3.8 μL RNase-free water and 0.7 µL dNTPs. The N1 gene primers mix involved in this study has been reported previously . The reaction was incubated at room temperature for 5 minutes to allow UDG enzyme to function before RT-LAMP assay. The amplification was carried out in heating block at 65°C for 60 minutes followed by deactivation at 80 o C, 2 minutes. Phenol red was added in the master mix for visual detection of end product by observing colour changes. Positive LAMP reaction turns yellow and negative reaction remained in pink (Figure 1) . Phenol red is a water-soluble dye that are widely been used as a pH indicator in various medical and cell biology tests. At high pH, the dye will show red colour while at low pH, it turns to yellow colour. During RT-LAMP amplification process, there are production of pyrophosphate and hydrogen 6 ion as by by-products from the incorporation of dNTP/dUTP by the polymerase, that causes pH drop from alkaline pH to a final acidic pH (Notomi et al. 2000) . Analytical sensitivity using 10-fold serially diluted in vitro transcript RNA with known numbers of nucleic acid copies showed the lowest detection limit as 1 copy/µL RNA; whereas analytical specificity using genomic RNA of coronaviruses Table 1 ). We believe that risk of carryover contamination could be reduced by incorporation of dUTP into the RT-LAMP assay. On top of that our previous study using carry-over simulations indicated that UDG can effectively prevent carryover contamination in the LAMP assay up to 0.6×10-13g of LAMP products (Zen et al., 2020) . Results revealed that UDG RT-LAMP took a longer time (~9 minutes) for the entire RT-LAMP amplification process compared to normal RT-7 LAMP with dNTP (Supplementary Table 1) . Also, addition of dUTP into the master mix may slightly affect the sensitivity of UDG RT-LAMP as 3 samples were not amplified successfully. To minimize cost, a novel RNA extraction method from nasopharyngeal swab samples was used in the present study. The commercial kit requires USD$6.45/reaction as compared to our newly developed RNA extraction method which costs only USD$2.27/reaction. Therefore, this simple RNA extraction method could be recommended as an alternative for laboratories experiencing shortage of extraction kits. We managed to develop a simple and cost effective RNA extraction from nasopharyngeal swab samples. Couple with additional of phenol red as indicator, this UDG RT-LAMP method is highly recommended to be used a diagnostic tool for diagnosing SARS-CoV-2 virus infection. Although dNTP RT-LAMP is slightly more sensitive than UDG RT-LAMP, dNTP RT-LAMP always has an issue of false positive result due to the carryover contamination problems. Our results showed that out of 40 negative rRT-PCR tested, 6 samples were amplified by dNTP-RT-LAMP. However, UDG RT-LAMP took a longer time for the amplification. The effectiveness of UDG-LAMP can be improved by further optimization of the ratio of dUTP and dNTP in the reaction mix (Kim et al., 2016) . As a comparison, rRT-PCR is a gold standard for the diagnosis of SARS-CoV-2, however, rRT-PCR took the longest time to complete the amplification among the three methods (90 mins). Moreover, the cost of rRT-PCR is expensive (~$40/reaction) as compared to RT-LAMP ($1.66). Simultaneous elimination of carryover contamination and detection of DNA with uracil-DNA-glycosylasesupplemented loop-mediated isothermal amplification (UDG-LAMP) Loop-mediated isothermal amplification for the detection of SARS-CoV-2 in saliva Uracil-DNA glycosylasetreated reverse transcription loop-mediated isothermal amplification for rapid detection of avian influenza virus preventing carry-over contamination Real-time reverse transcription loopmediated isothermal amplification for rapid detection of SARS-CoV-2 Loop-mediated isothermal amplification of DNA Evaluation of alternative RNA extraction methods for detection of SARS-CoV-2 in nasopharyngeal samples using the recommended CDC primer-probe set Advanced uracil DNA glycosylase-supplemented real-time reverse transcription loop-mediated isothermal amplification (UDG-rRT-LAMP) method for universal and specific detection of Tembusu virus Elimination of contamination in loop-mediated isothermal amplification assay for detection of human malaria This study was supported by Prototype Research Grant Scheme (PRGS), PR001-2020B (PRGS/2/2020/SKK09/UM/02/1) from the Ministry of Education, Malaysia.We acknowledge support of NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH for providing the following reagents; gammairradiated SARS-coronavirus, NR-9547. We would like to thank the Director General of Health Malaysia for his permission to publish this article. No conflict of interests declared. Ethical approvals for this study were obtained from UMMC Medical Ethics Ministry of Health Malaysia (NMRR-20-2344-56994).