key: cord-0806740-u889judu authors: Maiti, Biswajit; Anupama, Karanth Padyana; Rai, Praveen; Karunasagar, Indrani; Karunasagar, Iddya title: Isothermal amplification‐based assays for rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2: Opportunities and recent developments date: 2021-07-03 journal: Rev Med Virol DOI: 10.1002/rmv.2274 sha: fe96109a60da7a9ec31bb294e29474811a311e14 doc_id: 806740 cord_uid: u889judu The coronavirus disease 2019 (COVID‐19) is a global pandemic caused by a novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). To date, the virus has been detected in 219 countries of the world. Therefore, managing the disease becomes the priority, in which detecting the presence of the virus is a crucial step. Presently, real‐time RT polymerase chain reaction (RT‐qPCR) is considered a gold standard nucleic acid amplification test (NAAT). The test protocol of RT‐qPCR is complicated, places high demands on equipment, testing reagents, research personnel skills and is expensive. Therefore, simpler point‐of‐care (POC) tests are needed to accelerate clinical decision‐making and take some of the workload from centralized test laboratories. Various isothermal amplification‐based assays have been developed for the sensitive detection of different microorganisms, and recently some of them have been applied for detection of SARS‐CoV‐2. These do not require any programable thermocycler, can produce the results in a single temperature, and therefore, are considered simple. Unlike RT‐qPCR, these methods are highly sensitive, specific, less time‐consuming, simple and affordable, and can be used as POC diagnostic kit for COVID‐19. In this review, we have discussed the potential of isothermal amplification‐based assays as an alternative to RT‐qPCR for the detection of SARS‐CoV‐2. capacity. The availability of sophisticated testing facility in many districts of India is a serious limitation. Even in some of the developed countries, testing is limited to only symptomatic individuals. Hence asymptomatic carriers are not detected, and they could continue to spread the virus. To estimate the actual magnitude of the disease, a rapid and affordable diagnostic method is essential. Usually, the detection of RNA virus (causing acute respiratory infection, in particular) is carried out by probe-based real-time RT polymerase chain reaction (RT-qPCR). [1] [2] [3] [4] [5] [6] [7] The method has been well accepted, as it has a high level of sensitivity and can offer very low limit of detection (LOD). However, performing RT-qPCR requires sophisticated instruments, long reaction times, and due to the complexity associated with result analysis, skilled personnel are required. Furthermore, false-negative results at a late stage of the disease due to low viral load in the upper respiratory tract could be a problem. On the other hand, the serology tests for COVID-19 have also been recommended by the CDC, but this is an indirect method of detecting SARS-CoV-2, as it detects antibodies in patients' blood. 8 Hence, alternative rapid, portable, and affordable nucleic acid amplification tests (NAAT) are required for the detection of SARS-CoV-2. Unlike PCR-based methods, isothermal amplification-based assays are simple processes that rapidly amplify the nucleic acids at a constant temperature and do not require any programmable thermocycler. 9 These methods are well-accepted since they are highly sensitive, specific and simple over other molecular diagnostic techniques. During amplification, high pyrophosphate ion by-product levels are produced, which can change the pH of the reaction mixture. Therefore, a pH-sensitive dye can be used for simple visual detection. 10 Various isothermal amplification methods that can be used for the detection of SARS-CoV-2 are listed in Table 1 . Looking at these assays' potential, regulatory authority like US Food and Drug Administration has approved the use of kits based on isothermal nucleic acid amplification (https://www.fda.gov/media/138248/ download). Here we have reviewed the potential applications of isothermal amplification assays for the detection of SARS-CoV-2 to combat COVID-19. While various isothermal assays have been developed and used for nucleic acid amplification, loop-mediated isothermal amplification (LAMP) assay is relatively popular, developed by Notomi et al. 11 The isothermal amplification-based assays involve simple pre and postprocessing methods. The sample collection to completion of the test requires very little time compared to other molecular methods like qPCR. Therefore, these have the potential of a point-of-care (POC) diagnostic tool for detection of SARS-CoV-2. One of the important pre-requisites in any molecular diagnostic technique is the use of purified target nucleic acid. It is generally time-consuming and costintensive to obtain a relatively pure form of nucleic acid. However, in isothermal amplification-based assays, this pre-requisite can be done away with since crude cell lysate can be used as a template for the reaction. Moreover, the end products can be detected in various ways, such as visualization by the naked eye, using a turbidimeter, lateral flow dipstick, or agarose gel electrophoresis. On the other hand, these isothermal amplification-based assays usually provide only qualitative results and can be susceptible to contaminations and chances of generating false-positive results. Some of the isothermal amplification-based assays have been developed for the sensitive and accurate detection of SARS-CoV-2 (Table 2 ). An overall detection process of SARS-CoV-2 using different isothermal amplification-based assays was shown in Figure 1 . However, few of these methods have been optimised for the detection of a novel coronavirus, SARS-CoV-2 using specific genes such as RdRp, spike, E and nucleocapsid genes ( Figure 2 ). The updates on these methods for the detection of SARS-CoV-2 have been discussed here. LAMP assay is one of the most studied isothermal amplification assays and has been used to detect various microorganisms. Various investigators across the globe have been working on developing LAMP-based POC diagnostic tool for the detection of SARS-CoV-2. This method uses a single temperature for the amplification of nucleic acid. The method requires Bst polymerase enzyme with strand displacement and polymerization activity and four primers that bind six regions in the target during the amplification. 11 RT-LAMP assay is a variant of the conventional LAMP assay, uses RNA as a template, RT enzyme for the synthesis of cDNA, Bst polymerase and primers. Initially, the RT-LAMP was developed for the detection of West Nile Virus by Parida et al. 12 The RT-LAMP has been used as a diagnostic tool for different viruses, including SARS-CoV-2. Target genes for the detection of SARS-CoV-2 include orf1ab, nucleocapsid phosphoprotein (N), spike protein gene (S), envelope protein (E) gene, membrane glycoprotein gene. [13] [14] [15] [16] [17] Baek et al. 18 reported RT-LAMP as a rapid early-detection method for novel SARS-CoV-2, and the assay allows the detection of SARS-CoV-2 in 30 min with the detection limit of 10 2 RNA copies. A CRISPR-Cas12 combined LAMP assay was developed and found to be faster as compared to the Centres for Disease Control and Prevention (CDC) recommended real-time RT-PCR assay. The assay also showed 95% positive predictive agreement and 100% negative predictive agreement. 19 Zhang et al. 20 When swab samples are used in the validation of RT-LAMP assay, they found that when the viral load is high RT-LAMP assay is suitable to detect the infection but if there is low viral load sensitivity of RT-LAMP assay is not enough to detect SARS-CoV-2. 22 pandemic has shown us the importance of POC diagnostic tool in controlling the pandemic; hence RT-LAMP assay can be used in the field of diagnosis for the detection of SARS-CoV-2. The nucleic acid sequence-based amplification (NASBA) is an isothermal transcriptional based nucleic acid amplification assay, which is also known as 'self-sustained sequence replication' (3SR). 23 The NASBA is majorly used for the in vitro amplification of RNA in a short time. The assay requires two sets of primers and three different enzymes, including avian myeloblastosis virus (AMV) RT, RNase H and T7 DNA dependent RNA polymerase. 24, 25 The assay can generate 10 13 copies from an initial copy number of 10 4 in 2 hours. 23 Lu et al. 26 T A B L E 2 Comparison of isothermal amplification-based assays with other presently available methods for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) The recombinase polymerase amplification (RPA) was discovered in 2006 by Piepenburg et al. 47 It is a relatively simple isothermal assay that can be performed in a short period of time. 48, 49 In this method, recombinase enzyme plays an important role, hence the name RPA. Initially, recombinase enzyme forms a complex with primers and unwind the duplex DNA, later DNA polymerase enzyme displaces the strands and amplifies the nucleic acid. 43, 50 The nucleic acid-based technique has been carried out for the detection of various microorganisms. RPA assay for the detection of HIV-1 was developed as a rapid detection method without the use of any heating instrument, optimized in ambient temperature. 48 Huang et al. 51 developed RPA-CRISPER based detection method for the sensitive detection of SARS-CoV-2. The high-throughput CRISPR-p based RPA assay was able to deliver the results in approximately 50 min, and the detection limit was two copies per sample. This method has lot of potential to be a diagnostic tool for the detection of SARS-CoV-2. It's already been tried as diagnostic method by few researchers. However, more studies are required to validate and optimize the assay for simple end-point detections. Another isothermal amplification-based assay, rolling circle amplification (RCA), is one of the oldest isothermal assays developed in 1995. The assay is based on the in vitro replication of circular DNA. In this method, the circular form of nucleic acid serves as the tem- The sensitivity and specificity of the assay is a crucial point for any detection method to become successful and qualify as POC diagnostic tool. The RT-qPCR is considered a gold standard for detecting SARS-CoV-2, as it offers a high degree of sensitivity and specificity. Therefore, an alternative method like isothermal amplification-based 6 of 9assays must provide good sensitivity and specificity as compared to the gold standard. Among various isothermal amplification assays, more studies have been carried out using LAMP assay for SARS-CoV-2 detection. The assays are found to be highly sensitive, which is comparable with RT-qPCR assay. The study reports that isothermal amplification-based assay could detect one femtogram of the RNA, where RT-qPCR also demonstrated similar sensitivity. 14 Similarly, as low as 1-10 copies of RNA of SARS-CoV-2 virus can be detected by the isothermal assay. 56 Studies of Thi et al. 22 and Lu et al. 57 reported isothermal assays could detect 100 copies or less of RNA of SARS-CoV-2. However, the LOD can vary from one target gene to the other. In general, isothermal amplification-based assays were found to 80%-100% specific for the detection of SARS-CoV-2. 56 Similarly, the specificity of the isothermal amplification-based assays also been tested with the help of in silico analysis. For instance, Lamb et al. 14 The POC diagnosis should be rapid, simple and easy to handle. Various investigators have reported that isothermal amplificationbased assays like LAMP assay are much more straightforward and relatively quicker than the gold standard RT-qPCR. 58 The isothermal amplification-based assays can be performed in less than an hour, and results can be interpreted much faster than RT-qPCR. In general, isothermal assays are reported to be an easy and less time-consuming method for the detection of viral pathogen. 59 The isothermal amplification-based assays can be further simplified were not found to be successful in detecting multiple targets in a single reaction. 33 On few occasions, primer mispairing and amplification inhibition are some of the disadvantages which will result in non-specific amplification of the products. 63 NASBA assay has its disability, which requires an initial denaturation step before the actual amplification. Also, this method sometimes fails when the temperature is more than 41°C due to enzyme degradation. Additionally, it can only amplify the target, which is more than 100 bp. 23, 43 Some isothermal methods have restrictions over the type of templates used for amplification. For example, RCA method requires a circular DNA or RNA as a template. 33 Overall, if the isothermal amplification-based assays are optimised well, some of these limitations can be minimised and considered for the detection of SARS-CoV-2. COVID-19 testing based on the individuals showing symptoms of the disease is inadequate to identify asymptomatic carriers and develop containment strategy. Therefore, testing populations is important for diagnosis, epidemiology and surveillance. The isothermal amplification-based assays methods can be a good alternative for the sensitive detection of SARS-CoV-2. In a post-COVID pandemic situation, it will be important to monitor all patients coming into the hospital to differentiate SARS-CoV-2 positive patients to be kept in isolation wards. Therefore, various rapid isothermal amplification-based assays could be useful for this screening purpose. These methods can be delivered in a few hours, and the kits will find application for this purpose. Since the tests are based on isothermal amplification and visual detection, they can be performed in a simple lab set-up without the requirements of sophisticated instruments, which can be made available even at a rural primary health centre. This way, even people living in rural areas will have access to COVID-19 tests. 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The authors declare no conflicts of interest. The data that support the finding of this study are available from the corresponding author upon reasonable request. MAITI ET AL.