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Accurate LAMP-Enabled Rapid Test (ALERT) for COVID-19 detection: a need of the hour

Anita Sharma, Puran Parihar, Arvind Nanera, Sumeru Samanta, Neha Sharma, Sohil Takodara

Department of Biochemistry, HIMS Dehradun, Uttrakhand, India; Department of Pathology, Geetanjali Medical College & Hospital, Udaipur, Rajasthan, India; Clinical & Molecular Biology, SCSRD, Hazaribagh, Jharkhand; Department of Biochemistry, Rohilkhand Medical College, Bareilly, Uttar Pradesh, India; Department of Biochemistry, Geetanjali Medical College & Hospital, Udaipur, Rajasthan, India

COVID -19 virus has challenged many countries with another bigger and deadly upsurge. National health care infrastructure especially medical diagnostic facilities are under immense pressure. The pandemic has highlighted the cardinal importance of rapid and accurate diagnostic assays as rapid identification and isolation of infected individuals and tracing those who have been in contact with, is crucial in chain breaking and reducing the spread of COVID -19.

Despite the nationwide acceptance by central & state governments and medical councils, of PCR-based assays as a gold standard for molecular detection of COVID-19 infection, these diagnostic methods require skilled personnel and sophisticated infrastructure that makes them not viable in the setting of countries with limited resources like India. Moreover, these techniques are time-consuming and have complicated protocols that limit their diagnostic utility and efficacy.

Delay in results especially in rural areas with less developed health infrastructure in this current situation with an exponentially increasing number of new cases and in absence of confirmed laboratory results in further transmission.

Lack of a cost-effective, reliable testing method has worsened the scenario and an urgent need to switch to a rapid, sensitive, specific, and inexpensive point of care test (PoCT) in order to successfully identify the infected patients early, provide them necessary medical support and take necessary measures to control the spread of disease.

The present-day situation obliges an equally reliable molecular detection method for diagnosis and control of COVID-19. One-step loop-mediated isothermal amplification (LAMP) reaction is capable of detecting even a few copies of target nucleic acid sequences ounder isothermal conditions (usually 60–65C) with the help of specially designed primer sets. Due to its simplicity, this technique is attaining more acceptance in the diagnosis of viral diseases.

Loop Mediated Isothermal Amplification Technology 

Loop-mediated isothermal amplification or LAMP is one of the best suitable molecular assays for detection of target nucleotide sequence where amplification is rapid and takes place at a single temperature (isothermal)that can also be used for detection of RNA template for which additional reverse transcriptase enzyme is essential. (RT-LAMP)

LAMP fulfils “ASSURED” (affordable, sensitive, specific, user-friendly, rapid and reliable, deliverable to end-user) characteristics which makes this technique suitable for detection of various viral, bacterial, and other microbial infections, often directly from clinical samples.

Sample collection 

(i) Upper respiratory tract specimen – nasopharyngeal (NP) specimen, oropharyngeal (OP) specimen

(ii) Lower respiratory tract specimen- bronchoalveolar lavage or lower respiratory tract aspirates

(iii) Other clinical samples – blood, faeces[10,11] Reagents.

DNA polymerase, magnesium salts, deoxynucleotide triphosphates (dNTPs), heat-stable reverse transcriptase (for detection of RNA viruses), four to six primers including two loop primers, betaine, and buffer solution for the enzyme.

PROCEDURE

The sample is collect and transferred to a suitable container. Adsorbent tubes are used to remove inhibitors present in the sample that may interfere with the reaction. The samples or extracted viral nucleic acids were then mixed with the LAMP oreagents and heated at 60-65C for about 30 min to complete the amplification.

The process starts with the selection and development of disease-specific primers. There are various online tools with the help of which one can choose and design disease-specific primers also known as LAMP primers such as Primer Explorer and LAVA (LAMP Assay Versatile Analysis).[14] LAMP molecular assay is summarized in the following steps:

1. Identification of target sequence:Region of interest is identified. Here six genomic sequences are identified, three forward sequences (F1, F2, F3) and three backward sequences (B1, B2, B3). LAMP primer is now designed according to the target sequence. 

2. Denaturation of Nucleic acid sequence (DNA)

3. Activation of Forward Inner Primer (FIP): attaches on complimentary F2c region. FIPis a hybrid primer that also contains F1c complementary sequence (additionally). It starts adding complementary bases in 3′-5′ direction.

4. Forward outer primer (F3) primer Starts functioning: iv (a) Initiation of complementary nucleotide additionF3 primer attaches to its complementary region on template strand upstream to F2c-FIP binding region (iv-a). iv (b) Elongation of complementary strand – Primer F3 now unzips the complementary strand made by FIP primer & adds nucleotide according to template strand (iv-b). iv (c) Half Dumbbell like structure formation-now Strand made by FIP primer, which has a complementary sequence (F1c) for F1genomic sequence, it forms a half- dumbbell like structure by hybridizingon itself at 5’end

5. Activation of Backward Inner Primer (BIP): Attaches on B2c region of a newly synthesized strand (from FIP), BIP is a hybrid primer that additionally contains B1c complementary sequence (which is responsible for loop formation later on). It starts adding complementary bases in 3′-5′ direction.

6. Backward outer primer (B3) primer starts functioning: vi (a) Initiation of complementary nucleotide additionB3 primer attaches to its complementary region on template strand upstream to B2c-BIP binding region (Fig. vi-a). vi (b) Elongation of complementary strand- Primer B3 now unzips the new strand formed by the action of BIP and synthesize a new strand accordingly (Fig vi-b). vi (c) Half dumbbell-like structure formation-The strand synthesizes by BIP primer now forms a half- dumbbell structure, as it has two complementary sequences one is B1c on BIP primer and another is B1 on the newly synthesized strand.

7. Complete dumbbell structure formation: Asthe strand synthesizes by FIP and BIP primer contains two complementary sequences (F1/F1c & B1 /B1c), these sequence binds with each other in the newly synthesized strand and form two-loop structure (dumbbell)

ANALYSIS OF RESULTS 

The LAMP assay rapidly (within an hour) amplifies and produces a large number of DNA copies, usually 100 times higher than conventional PCR.

Detection of amplification of the target sequences can be assessed by

1. Either a simple visual examination of the turbidity that results from white precipitation of magnesium pyrophosphate as a by-product of the reaction or using an inexpensive turbidimeter to quantify the turbidity at 650 nm.

2. Agarose gel electrophoresis [16]

3. Dye-based fluorescence assay (DNA intercalating agents such as SYBR Green I). [17]

4. A colorimetric visual inspection method [18] in which Amplicons mixed with the dye and the colour change indicates the presence of a positive reaction.

However, for the sake of rapidity, visual detection can be an immediate and straightforward option for laboratories without specialized equipment in the context of COVID-19 diagnosis. A diagrammatic representation of the LAMP technique from sample collection to analysis of results.

LAMP versus Immunoassay 

Immunoassay is of much importance in epidemiological studies and vaccine development, these methods are based on either detection of antibodies of disease-specific antigen in serum or antigen detection in plasma or other body fluids.

A. Immunological assays are useful in the detection of IgM which is an indicator of early-stage infection or short-term immunity and (days to weeks) or detection of IgG levels, which reveals long-term immunity (years or permanence)and presence of post-infection immunity. Prevailing antibody detection methods include traditional enzyme-linked immunosorbent assay (ELISA), immune chromatographic lateral flow assay, neutralization bioassay, and specific chemosensors.

B. Another variant of immunoassay is the Antigen Detection method, which utilizes antibodies to detect antigen(s) in serological samples.

Though immunoassays have inherent advantages of simple handling, cost-effectiveness, less turnaround time due to automation but they are associated with disadvantages of the requirement of dedicated laboratories and staff, less accuracy, sensitivity, and specificity of lab results.

LAMP versus RT -PCR 

Though the traditional method of RT PCR is widely accepted and used in the detection of viral, bacterial, and other pathogens, the loop-mediated isothermal amplification method of the molecular assay also shows comparable specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%).

Loop-mediated isothermal amplification (LAMP) reaction is a novel nucleic acid amplification technique that not only overcomes the timeconsuming and laborious procedure of the traditional RT-PCR technique but also has many advantages over it. Point of care testing: As there is no requirement of sophisticated instruments, LAMP molecular assay can be functional as a portable device, which can be used in an emergency setting and hospital ward setting as POCT devices for quick and reliable results.

Robust and less turnaround time: LAMP is a robust technique, there is no need for stringent preparation, more than a hundred times amplification products are produced in less than one hour, and even matrix inhibitors don’t affect the results.

Cost-effectiveand storage requirements: Reagents are oto be stored at 4C (ordinary fridge), whereas the PCR-based reagents are to be stored and transported at o–20Cwhich needs deep freezers that escalate the cost . [24,25,26,27]

Clinical applications: 

Diagnosis of Human Diseases 

LAMP diagnostic methods are useful for the diagnosis of various human diseases

Bacterial pathogen 

Mycobacterium Tuberculosis and Mycobacterium species (M. intracellulare, M. marinum, M. kansasii, M. avium, M. flavescens, M. smegmatis, M. fortuitum)

Bacterial pathogen such as Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae)

Viral pathogen 

Human immunodeficiency virus (HIV)[33], Japanese Encephalitis virus [34], Human papillomavirus [35], Dengue virus [36], West Nile virus [37], Mumps virus [38], Highly pathogenic avian influenza (HPAI) H5N1 virus [39,40], SARS virus [41], MERS virus [42] and Chikungunya virus.

Plasmodium Detection

This technique is useful in the detection of different species of Plasmodium (Plasmodium vivax, Plasmodium falciparum, and Plasmodium malariae andPlasmodium knowlesi and Plasmodium ovale) with 100% sensitivity and 100% specificity by targeting the 18S rRNA gene of plasmodium.

Early detection of cancer

LAMP is useful in the early and confirmatory diagnosis of malignancy. This technique is also helpful in differentiating the chemo-sensitivity of the malignant tumour.

Advancements in LAMP based techniques for the diagnosis of COVID-19 

Covid -19 is a positive-sense, single-stranded RNA (+ssRNA) virus. The viral genome is made up of about 30000 nucleotides containing one structural gene unit and a non-structural gene unit. Structural gene unit encodes for nucleocapsid (N) protein, envelop (E), membrane (M), and spike (S) proteins, out of which spike glycoprotein is responsible for the interaction with the host cells and two large, open reading frame genes (ORF1a and ORF1b) that codes for sixteen non-structural proteins (NSP) including RNA-dependent RNA polymerase (RdRp) [47–51]. Diagrammatic representation of COVID-19 genome shown in fig.3 

Based on information of genomic sequence, extensive work and developments are going on related to lampbased techniques for rapid and accurate detection of COVID -19. These methods based on targeting the different gene of the viral genome are summarized in table-3.

CHALLENGES AND LIMITATIONS

Molecular assays are sensitive, specific, and reliable, and confirmatory, however, traditional techniques such as RT-PCR are sophisticated, complex, expensive, and time-consuming. LAMP is an emerging molecular technique that minimizes most of these problems but there are certain limitations that can be categorized as pre-analytical and analytical.

Pre-analytical 

1. Sample collection, transport, and storage: As with other molecular assays, skilled and trained personal is required for proper sample processing. Results may be erroneous due to inappropriate sample collection, cross-contamination of the sample, storage, transfer, and processing of the sample.

2. Different viral load in different sample & diagnostic timing: Samples are usually collected from upper and lower respiratory passages such as nasal, nasopharyngeal, oropharyngeal swabs, sputum, lower respiratory tract aspirates, broncho alveolar lavage fluid, and nasopharyngeal wash/aspirate or nasal aspirate. However, the biodistribution of virus particles may vary in different specimens, which can result in falsenegative results. [61] The knowledge and understanding of the detectable SARS-CoV-2 load in the respiratory tract during different stages of infection is still evolving [62]. The viral load kinetics also varies from person to person and depends on several factors including the patients’ epidemiological history, immune response, and treatment or medication effects [63]

3. Mutations in viral genome : Mutation through genetic recombination as well as genetic diversity ofSARS-COV-2 presents with more challenges for LAMP molecular assays. Osório and CorreiaNeves reported that~79% (26/33) primer binding sequences in at least one gene used in molecular assayswere mutated compared to Wuhan-Hu-1 strain-NC 045512 [64]

4. Development and designing appropriate primer: The main hindrance of the LAMP-based method is the intricacy of primer design

Continue optimization of the oligonucleotides primer through frequent analysis of the updated genomic sequences is essential to enhance their accuracy and reliability.

Analytical

1. Procedural: Due to the carry-over from the previous sample leads to false-positive results. However, automation and use of fluorescent primers may minimize this problem

2. Quality assurance: Quality assurance programme is the heart of functioning of any diagnostic laboratory, intending to maintain accuracy and precision of lab results. As LAMP is an emerging technique, commercial availability, and accessibility of high-quality control material (standards /controls/calibrators) is challenging.

3. Biosafety: Working with a virus such as COVID -19 require a diagnostic laboratory with Biosafety Level 3 (BSL-3). Test verification requires the use of appropriate biosafety and protective equipment while handling, manipulating, and testing specimens. There must be strict compliance with Good Laboratory Practices during all stages of SARS-CoV-2 diagnostic molecular testing with the use of appropriate disinfectants, decontamination of work surfaces and equipment [67, 68]

4. Biohazard risk management system: There should be proper handling of all biohazard material.

Conclusion and perspectives 

In the currentpandemic situation, there is a need of a real-time method besides prevailing RT PCR testing, LAMP is confirmatory, precise, cost-effective, quick, user-friendly molecular diagnostic method, which is essential to address the COVID-19 pandemic.

LAMP technology promises various advantages in curtails the present pandemic situation. In developed nations of the world, this technique is already in mainstream diagnostic facilities. The RT-LAMP technology is perfect for our current situation. There is a need for urgent approval, clinical verification, validation, certification, and training for timely commencement of this ideal diagnostic technique for curtails this dreadful health emergency.

Proper evaluation, assessment, and audit of the prevailing diagnostic tests should be done, to identify their diagnostic accuracy, efficiency, and errors.

These approaches will be beneficial for further planning and COVID-19 management and improve the future readiness for emerging infectious diseases.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Ethical conduct of research

The authors declare that this review article does not require Institutional Review Board/Ethics review or approval.

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