PMID 41054873 | Advances in Isothermal Amplification for the Diagnosis of Tuberculosis.
DOI 10.1002/jcla.70113 | Journal of clinical laboratory analysis 2025
PMCID PMC12618186

 3636 jclinlab Journal of Clinical Laboratory Analysis J Clin Lab Anal Wiley PMC12618186 12618186 12618186 41054873 10.1002/jcla.70113 Advances in Isothermal Amplification for the Diagnosis of Tuberculosis Jirakittiwut Nuttapon 1 Ratthawongjirakul Panan 1 ✉ 1 Center of Excellence for Innovative Diagnosis of Antimicrobial Resistance, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand * Correspondence: Panan Ratthawongjirakul ( panan.p@chula.ac.th ; panan_etc@yahoo.com ) ✉ Corresponding author. 7 10 2025 39 21 e70113 e70113 16 11 2025 © 2025 The Author(s). Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made. ABSTRACT Background Detecting tuberculosis (TB) remains challenging in low‐resource settings due to limited access to advanced diagnostic tools. The emergence of isothermal amplification techniques, such as loop‐mediated isothermal amplification (LAMP), presents a promising solution for TB detection. Several isothermal amplification techniques, e.g., strand displacement amplification (SDA), helicase‐dependent amplification (HDA), recombinase polymerase amplification (RPA), and rolling cycle amplification (RCA), have been attracting attention for development as point‐of‐care testing (POCT) in many diagnostic fields. The present review summarizes the most common isothermal amplification techniques for the detection of TB. Methods To collect the necessary information, we searched PubMed and Scopus for scientific evidence published from 2000 to September 2024, using the keywords loop‐mediated isothermal amplification tuberculosis, strand displacement amplification tuberculosis, helicase‐dependent amplification tuberculosis, recombinase polymerase amplification tuberculosis, rolling circle amplification tuberculosis, polymerase spiral reaction tuberculosis, cross‐priming amplification tuberculosis, and multiple cross displacement amplification tuberculosis. Results The methodologies of the most usual isothermal amplification techniques are addressed, as well as the advantages and limitations of the technique, highlighting applications in TB diagnosis. Exploring these advances addresses the principles of innovation in isothermal amplification, including their performance and applicability across diverse sample types. Some techniques have already been launched into routine TB diagnosis, while other promising tests are still being researched or evaluated. These techniques are easy to perform and provide results rapidly, enabling prompt treatment within a few hours. Isothermal amplification techniques may be one of the key molecular tools to fight TB in pursuit of the goals of the End TB strategy. Keywords: IS6110, isothermal amplification, Mycobacterium tuberculosis complex, tuberculosisIS1081 The emergence of isothermal amplification techniques presents a promising solution for tuberculosis (TB) diagnosis. These methods are attractive due to their simplicity, speed, and ability to perform without specialised equipment, which could make them suitable for low‐resource settings. Therefore, isothermal amplification techniques may be one of the key molecular tools used to fight TB in pursuit of the goals of the End TB strategy. status released display-pdf yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Revised 2025 May 26; Received 2024 Dec 6; Accepted 2025 Sep 26; Collection date 2025 Nov. 1. Introduction Epidemiological studies show that Tuberculosis (TB), caused by the Mycobacterium tuberculosis complex (MTB), is a major global health problem, especially in low‐ to middle‐income countries. The World Health Organisation (WHO) reported that TB case detection greatly decreased after the COVID‐19 outbreak in 2020. However, TB case notifications also significantly increased, especially in Africa, the Eastern Mediterranean, America, and Southeast Asia, when the COVID‐19 crisis receded in 2021 [ 1 ]. Thus, accurate and rapid diagnosis is critical for controlling TB. Once the suspected TB patient is identified, appropriate and prompt treatment can be performed, thereby improving patient retention, preventing TB spread, and reducing the TB epidemic. A decline in the global TB incidence rate to achieve the End TB Strategy goals depends on a rapid and accurate diagnostic method. Molecular biological technology for the diagnosis of TB has been continuously developed due to its short time consumption with high accuracy, sensitivity, and specificity efficiency. Several developed methods or test kits based on molecular biology have been utilised to diagnose TB. The technique can detect a small amount of pathogens obtained from either culture assays or direct specimens and is adaptable for diverse applications. However, the high‐cost instrumentation, test cost, and equipment maintenance limit these methods, especially in low‐income countries or for point‐of‐care testing (POCT). There is an urgent need for tests that are deployable at rural TB laboratories at a community level, where only basic laboratory infrastructure is available. Improved POCT TB diagnostic tests are meant to be implemented in decentralised laboratories where the patients make initial contact with the health system. The more quickly and widely TB patients are identified and properly treated, the more TB incidence may be reduced. Isothermal amplification technologies have been developed by mimicking in vivo amplification mechanisms. The reaction is performed at a single constant temperature without temperature cycling requirements for specific amplification. Advances in isothermal amplification technologies, which use only a simple heating box, a water bath or a basic temperature controller, could lead to a diagnostic tool for the POCT‐based screening test of TB without the need for specialized instruments. Since the 1990s, over 15 types of isothermal amplification techniques have been described, and a thousand studies related to the isothermal amplification of nucleic acids have been published. However, only a few techniques are approved for clinical diagnosis, while some methods have yet to be developed beyond proof of concept or academic research. The main objective of this review is to summarise the most commonly used isothermal amplification techniques for TB diagnosis, with a focus on their potential for use in point‐of‐care testing (POCT), particularly in low‐resource or decentralised settings. Only peer‐reviewed papers were included in this review, based on a comprehensive examination of their titles, abstracts, and full texts. Exclusion criteria were rigorously applied to eliminate irrelevant studies, including modelling studies, duplicate publications, editorials, guidelines, and studies with insufficient data. This is not a comprehensive review of all commercial isothermal amplification techniques; the study presents only the techniques used to detect MTB in clinical specimens. Some are already incorporated into routine diagnostics, while other promising tests are still being evaluated. 2. Isothermal Amplification for TB Detection 2.1. Loop‐Mediated Isothermal Amplification (LAMP) Since 2000, when LAMP was first introduced by Notomi et al. [ 2 ], the assay has become one of the most popular molecular techniques used in several study fields, such as medicine, agriculture, and the food industry. LAMP exhibits excellent performance in both sensitivity and specificity by using 4–6 target‐specific primers to recognise six specific sites flanking the amplified DNA sequence. The amplification process relies on only the strand‐displacement activity of a DNA polymerase and generates 10 9 copies of DNA from a few copies of template in less than 1 h at 60°C–65°C [ 2 ]. The Bst DNA polymerase enzyme is commonly used in LAMP and is resistant to the presence of anticoagulants, hemin, N‐acetylcysteine, sodium chloride and other PCR inhibitors [ 3 ]. The robustness of LAMP as a rapid and sensitive technique with satisfactory specificity has been shown in various works with remarkable results compared to those of other existing isothermal amplification techniques. Among the publications concerning TB using isothermal amplification during 2000–2024 (obtained from PubMed and Scopus), LAMP was used in 66%. The percentage was calculated by dividing the number of publications using LAMP by the total number of TB detection‐related publications employing isothermal amplification during 2000–2024. Recombinase polymerase amplification (RPA), helicase‐dependent amplification (HDA), strand displacement amplification (SDA), rolling circle amplification (RCA), and the remainder including polymerase spiral reaction (PSR), cross‐priming amplification (CPA) and multiple cross displacement amplification (MCDA) were used in 9%, 2%, 4%, 10%, and 8%, respectively (Figure 1 ). FIGURE 1 Number per year of scientific publications regarding amplification systems from 2000 until 2024. Data were searched by using the keywords “strand displacement amplification tuberculosis”, “loop‐mediated isothermal amplification tuberculosis”, “helicase‐dependent amplification tuberculosis”, “recombinase polymerase amplification tuberculosis”, “rolling circle amplification tuberculosis”, “polymerase spiral reaction tuberculosis”, “cross‐priming amplification tuberculosis” and “multiple cross displacement amplification tuberculosis”. All data were obtained from PubMed and Scopus (accessed in September 2024). LAMP‐based detection for MTB has been thoroughly developed to improve test performance. In 2003, the gyrB gene was the first mycobacteria‐specific LAMP target developed [ 4 ]. Since then, several other specific target genes, such as 16S rRNA [ 5 ], rimM [ 6 ], IS6110 [ 7 ], hspX [ 8 ], mpb 64 [ 9 ], sdaA [ 10 ] and IS1081 [ 11 ], have been used as targeted regions for amplification in MTB detection by the LAMP assay (Table 1 ). Simple detection methods, such as turbidity [ 5 , 33 ] or fluorescence detection methods [ 6 , 34 ], colorimetric detection [ 2 , 6 ], lateral flow assays [ 35 , 36 ], and electrochemical analysis [ 37 , 38 ], were employed as end‐point or real‐time detection platforms with LAMP reactions. However, probe‐based detection methods are not commonly used because they are not compatible with the cauliflower‐like structures of LAMP products [ 39 ]. Single‐copy targets, including gyrB , rimM , mpb 64, and sdaA , have been used to detect MTB using LAMP reaction combined with SYBR Green end‐point detection. These developments allow rapid and accurate identification of MTB with no cross‐reaction with NTM and other bacterial species in culture isolates and/or clinical specimens. The reaction time ranged from 35 to 60 min at 63°C to 65°C with a satisfactory limit of detection (1–200 copies) [ 4 , 6 , 9 , 10 ]. Efficient DNA amplification by MTB‐LAMP targeting 16S rRNA was observed in MTB, while giving a negative result for all tested M. avium complex and M. kansasii clinical isolates and other bacterial species [ 5 , 8 ]. When exploring spiked sputum, the sensitivity of the hspX LAMP assay appears to be sufficient to detect MTB, with a detection limit of 500 bacilli/mL of sputum (equivalent to 10 copies of DNA/reaction) [ 40 ]. TABLE 1 Summary of published isothermal amplification techniques for TB detection and their performances. Amplification techniques Target gene Sample type Temp (°C) Time (min) Detection LOD a Sensitivity Specificity References LAMP gyrB Sputum 63 60 Naked eye/SYBR 5–50 copies — b — [ 4 ] Culture colony 35 16 s rRNA Culture colony 64 60 Real‐time turbidimeter 20 copies — — [ 5 ] Sputum 10 CFU 100% 94.20% rimM Culture colony 65 60 Naked eye/fluorescent metal indicator 200 copies — — [ 6 ] IS6110 Culture colony 65 75–90 Naked eye/SYBR and turbidity 1 copy — — [ 7 ] agarose gel electrophoresis hspX Culture colony 67 < 60 Real‐time turbidimeter 10 copies — — [ 8 ] Spiked sputum 500 copies mpb 64 Intraocular samples 65 60 Naked eye/SYBR 2 copies 100% 85.70% [ 9 ] sdaA Sputum 65 45 Naked eye/SYBR 1 copy 100% 97.20% [ 10 ] Culture colony 63 45 Naked eye/SYBR 1 copy [ 11 ] Agarose gel electrophoresis IS1081 Culture colony 63 45 Naked eye/SYBR 2 copies — — [ 11 ] Agarose gel electrophoresis Culture colony 65 60–90 Naked eye/SYBR 200 copies [ 12 ] Agarose gel electrophoresis Sputum 65 60–90 Naked eye/SYBR 20 copies 100% 75.75% SDA IS6110 Culture colony 37 120 Chemiluminescent assay 0–25 copies — — [ 13 ] IS6110/16 s rRNA Culture colony 40 120 Electrophoresis 5 copies — — [ 14 ] IS6110 Respiratory specimens (e.g., BAL c , sputum, bronchial wash) N/A 120–180 BDProbeTec ET — 94.50% 99.60% [ 15 ] Extrapulmonary specimens (e.g., gastric aspirate, urine, sterile body fluid, pus, biopsy) — 92.30% 100% IS6110 Tissue samples 37 15 BDProbeTec ET — 31.50% 97.50% [ 16 ] IS6111 Synthetic DNA 52 120 Magnetic bead combined with probes — [ 17 ] IS6110 Clinical specimens 41 120 Chemiluminescent assay — 97.20% 96.10% [ 18 ] HDA IS6110 H37rv DNA 65 60 Single‐step protocol combining disinfection, thermal lysis and HDA—4% Gel agarose N/A — — [ 19 ] IS6110 H37rv DNA 65 < 30 A lateral flow device 2 copies — — [ 20 ] IS6110 Spiked oral swabs — 50 Fluorescence signal in Tiny Isothermal Nucleic acid quantification sYstem 2.5 × 10 5 copies — — [ 21 ] IS6110 Clinical specimens 65 90 Electrochemical genomagnetic assay 15 copies — — [ 22 ] IS6110 Synthetic oligonucleotide/clinical specimens 65 90 Electrochemical genomagnetic assay 15 copies — — [ 23 ] RPA IS6110 Culture colony 39 20 Fluorescence probes 6.25 fg (~1 copy) — — [ 24 ] IS1081 20 fg (~4 copies) — — IS6110 Sputum, respiratory specimen 39 20 Fluorescence probes — 87.50% 95.4% (culture) IS1081 — 91.40% 100% (culture) IS1081 Culture colony 39 30 Lateral flow strip 25 fg (~5 copies) — — [ 25 ] Sputum, respiratory specimen 100% 97.92% (culture) IS6110 Culture colony 37 15 SYBR Green I 0.5 ng 97.95% 100% (PCR) [ 26 ] IS1081 0.05 ng 99.32% 100% (PCR) mts90 Plasmid DNA 39 20 Fluorescent probes 6 copies — — [ 27 ] Sputum/BAL/lung tissues — 96.43% coincidence rate (culture) Ag85B Plasmid DNA 39 20 Fluorescent probes 4 copies — — [ 28 ] Sputum — 90% 98% (smear) RCA Ag85B Culture colony 37 180 Hybridization‐based assays using capture probe‐conjugated beads and fluorescent‐labeled reporter probe 1000 CFU/mL — — [ 29 ] N/A Synthetic DNA 30 120 Gold nanoparticle counting 10 fM — — [ 30 ] IS6110 Sputum 60 80 Electrophoresis 200 CFU/ml 77.20% 98.60% [ 31 ] IS6111 Plasmid DNA 30 80 Smartphone‐assisted fluorescent analysis combining with highly sensitive fluorescent copper nanoprobes 5 fg/μL — — [ 32 ] a Limit of detection. b Not indicated. c Bronchial alveolar lavage. IS6110 has been commonly used as an MTB‐specific target in numerous existing nucleic acid amplification assays. The amplification of IS6110 by LAMP assay for TB diagnosis was first proposed in 2010 [ 7 ]. This IS6110‐LAMP assay's analytical specificity was 100%, as positive reactions were obtained only with DNA belonging to MTB. At the same time, the assay's sensitivity was 1 copy, which was 20 times more sensitive than IS6110‐based conventional PCR. When testing with clinical sputum, the LAMP assay could detect all positive sputum samples, including low‐score sputum, which was difficult to detect by conventional PCR. IS6110 is an insertion sequence specific to the members of MTB with more than 99% genetic homology. Moreover, most members of MTB, including M. tuberculosis , contain multiple copies of IS6110 (0 to 25 repeats per genome) [ 41 ]. Therefore, this target should enhance sensitivity when used in molecular diagnosis for TB. A previous study using conventional PCR suggested that the IS6110 target was more sensitive than a single‐copy internal transcribed spacer (ITS) target when identifying MTB in paraffin‐embedded tissue [ 42 ]. However, a significant number of MTB strains with low copies [ 43 ] or no copies of the IS6110 gene element have been reported [ 44 , 45 ], which may disable the validity of molecular TB diagnosis. Nevertheless, IS6110‐based identification remains the internationally accepted standard. The region where a high proportion of no copies of the IS6110 strains has been reported should be considered for careful interpretation. Another putative insertion sequence, IS1081, was introduced into the LAMP assay for MTB detection 1 year after IS6110 [ 12 ]. IS1081 is carried by MTB strains with a stable copy number of 5–7 repeats per genome [ 46 ]. IS1081‐based LAMP exhibits excellent sensitivity and specificity in diagnosing TB. The extension of an incubation period from 60 to 90 min significantly improved the assay sensitivity from 72.2% to 100% [ 12 ]. Furthermore, a recent study revealed that IS1081 is useful to incorporate into the LAMP setup to include more members of MTB and to ensure the detection of IS6110‐lacking strains [ 11 ]. The commercial amplification kits for TB diagnosis based on the LAMP method have been certified and marketed, known as Loopamp, by Eiken Chemical Co. Ltd. (Japan) [ 47 ]. LAMP has been extensively developed for MTB diagnosis owing to its simplicity, accuracy, robustness and affordable cost and is suitable for use in high‐burden and resource‐limited settings. In 2016, the World Health Organisation (WHO) recommended the TB‐LAMP kit as a replacement test for sputum smear microscopy for diagnosing pulmonary TB in adults with signs and symptoms consistent with TB [ 48 ]. The kit is a manual assay that requires less than 60 min to perform and can be easily read with the naked eye under ultraviolet light (green fluorescence) or real‐time turbidity and costs approximately $6 per reaction (the WHO target price) [ 49 ]. The diagnostic accuracy studies performed in peripheral settings showed that the sensitivity of TB‐LAMP ranged from 92.1% to 100% in smear‐positive sputum compared to the bacterial culture. In the case of smear‐negative sputum, LAMP had lower sensitivity ranging from 51.2% to 90.3%. However, the specificity of TB‐LAMP in overall sputum ranged from 96.6% to 100% [ 50 ]. For extrapulmonary tuberculosis, a recent study indicated that the sensitivity and specificity of LAMP against culture and Xpert MTB/RIF were 85.7% and 88.9% and 88% and 86.7%, respectively [ 51 ]. Moreover, high performance of TB‐LAMP in persons living with human immunodeficiency virus (HIV)‐infected patients was observed, where sensitivity and specificity were in the range of 63.8% to 73.4% and 95% to 98.8%, respectively [ 48 ]. The limit of detection of TB‐LAMP was confirmed by spiking serial 10‐fold dilutions of known concentrations of MTB in pooled MTB‐negative sputa, and the result was 130 CFU/mL [ 52 ], which is similar to that of Xpert MTB/RIF (131 CFU/mL) [ 53 ]. One major disadvantage of LAMP is its complicated primer design. LAMP primers can be challenging to design manually [ 47 ]; thus, software programs are strongly recommended for ease of creation and the possibility of reaction success. Moreover, LAMP carryover contamination often leads to false‐positive results and needs to be addressed when LAMP is applied. However, a previous study successfully managed the contamination risk by completely replacing dTTP with dUTP in the sdaA LAMP reaction mix without adversely affecting amplification efficiency for MTB detection [ 10 ]. Furthermore, false‐positive results of LAMP can occur due to its heavy reliance on indirect detection methods such as turbidity and nonspecific dyes. Thus, more specific detection platforms, such as probe‐based assays, should be considered to improve the specificity of LAMP. 2.2. Strand Displacement Amplification (SDA) Walker et al. first proposed SDA in 1992 [ 54 ]. SDA integrates the action of two core enzymes, an endonuclease and an exonuclease‐deficient DNA polymerase, with four primers, generating approximately 10 8 copies of the DNA product in 2–2.5 h at 30°C–55°C [ 47 ]. Although the reaction was performed at a constant temperature, thermal denaturation (95°C) was necessary to initiate the reaction. SDA targeting IS6110 has been shown to amplify MTB DNA in a combination of various detection platforms [ 13 , 14 , 15 , 16 , 17 , 18 ] (Table 1 ). Coamplification in a multiplex SDA reaction using IS6110 specific to members of the MTB and 16S rRNA specific to the most clinically relevant species of mycobacteria was successfully developed, demonstrated reasonable specificity to MTB with no cross‐reaction to NTM and other bacterial groups [ 14 ]. SDA was used in conjunction with a commercial platform (BDProbeTec ET, BD Biosciences), providing a direct detection assay for the detection of MTB in clinical specimens [ 15 , 16 ]. This semiautomated walk‐away system combines SDA with a fluorescent‐energy transfer detection system in a closed and high‐throughput assay format. The percent sensitivity and specificity of BDProbeTec ET were 35.7%–92.3% and 97.5%–100%, respectively, for extrapulmonary specimens, while the corresponding values for respiratory specimens were 94.5% and 99.6%, respectively [ 15 , 16 ]. Although this method has high specificity and sensitivity, the need for a costly analyser for amplicon detection is a disadvantage. Despite the advantages of the SDA reaction, the low sensitivity of the SDA assay remains a primary concern. The decreased sensitivity of SDA may be due to the relatively low temperature at which the assay is carried out (between 30°C and 55°C) [ 47 ], making it prone to nonspecific primer binding that might cause nonspecific amplification, especially when the target is absent or present at a low copy number compared with the background DNA. However, the lowest detectable level of MTB is very low (~5 genome copies) due to the use of a multicopy target (~10 copies) of the IS6110 insertion element [ 14 ]. Recently, a simple, rapid, and sensitive method for MTB‐specific DNA detection was constructed based on SDA and magnetic beads (MBs). Combining SDA with the superior separation capability of the MBs‐Hp1 probe shows high accuracy and good potential for diagnosing TB. The method also distinguished a one‐base mismatch from MTB‐specific DNA, showing great promise for MTB genome single‐base polymorphism analysis [ 17 ]. 2.3. Helicase‐Dependent Amplification (HDA) HDA is a simple isothermal amplification that simulates the in vivo process of DNA replication since a helicase is used to separate dsDNA instead of heat. This method was developed by Vincent et al. in 2004 [ 55 ]. HDA is compatible with a wide range of detection systems, such as end‐point detection, real‐time detection, and lateral flow devices [ 47 ]. The expansion of HDA for the detection of MTB has been reported in which the result interpretation was performed with agarose gel electrophoresis, lateral flow assay, fluorescence signal, or electrochemical detection. Shetty et al. successfully combined the disinfection, lysis and TB‐HDA protocols into a single tube for one step at 65°C for 60 min, requiring no further intervention [ 19 ]. The greyscale pixel intensity of the gel agarose electrophoresis obtained from the one‐step reaction was equivalent to that obtained with a pure DNA control, suggesting that the integration of combined disinfection, lysis and HDA into a single tube for a one‐step reaction may simplify the sample preparation and diagnosis of MTB [ 19 ]. Several published HDA methods require at least 1 h of amplification time. However, Motre et al. optimised primer concentration, molecular crowding reagents and amplification enzymes to improve HDA amplification speed [ 20 ]. They successfully developed a speed‐enhancing HDA method in which the amplification time was significantly reduced from the original 60 min to less than 30 min with a limit of detection of two copies of genomic DNA [ 20 ]. In 2021, the portable isothermal amplification‐based platform Tiny Isothermal Nucleic acid quantification sYstem (TINY) was used for the qualitative real‐time detection of MTB via the HDA method. HDA combined with TINY requires less than 50 min until a threshold time, and the detection limit is 2.5 × 10 5 copies of IS6110 target [ 21 ]. One more advantage is that HDA does not require heat denaturation for the template. However, HDA still needs to improve on sample contamination and false‐positives due to the use of a constant temperature, leading to easy misinterpretation. In contrast, false‐negative results may occur because of test inhibition, particularly in complex biological matrices. Some of the oligonucleotide primer properties should be designed to achieve target‐specific amplification for the isothermal technique, e.g., primer size ranging from 24 to 33 nucleotides, melting temperature (Tm) from 60°C to 74°C, and GC% within the interval 35%–60% [ 56 ]. Primers designed for PCR amplification may be modified to reach the recommended Tm needed for HDA. The addition of a customised 22‐nt tail at the 5′‐end of the oligonucleotide primer can increase the Tm after the first amplification and the amplicon length, which is critical in this isothermal technique. Under these conditions, the sensitivity and selectivity of HDA are improved [ 57 ]. 2.4. Recombinase Polymerase Amplification (RPA) RPA was first introduced in 2006 by Piepenburg et al. and is currently commercialised under the licence of TwistDx Limited (Cambridge, UK) [ 58 ]. A few years later, it has become one of the most robust isothermal amplification techniques in many research areas, including pathogen detection and diagnosis. The two core enzymes, recombinase and DNA polymerase, and one protein, single‐stranded DNA binding protein (SSB), are required for the reaction, which proceeds at low temperatures (37°C–42°C) without an initial denaturation step [ 59 ]. The RPA reaction time ranges between < 10 and 40 min, making RPA the fastest nucleic acid amplification technique available [ 47 ], and the limit of detection is only 1 copy [ 60 ]. Thus, RPA has become a new‐wave amplification‐based method enabling many breakthrough studies in molecular fields. RPA is compatible with multiplexing, leading to the possibility of development as a “one‐pot” assay for multiple target detection [ 61 ]. RPA generates dsDNA products that can be easily detected by simple techniques. Several detection platforms combined with RPA allow different formats that are suitable for rapid molecular diagnosis, improved sensitivity, reduced manual handling, and improved cost‐effectiveness, such as agarose gel electrophoresis, fluorescence dyes or probes, lateral flow strips, solid phases, capillary electrophoresis, microfluidics, and microarrays (Table 1 ). RPA does not require an initial template denaturation step and is tolerant to common PCR inhibitors [ 47 ], making it suitable for development as a diagnostic kit for direct specimens. In addition, the resilience of RPA dried pellets permits cold‐chain‐independent transportation and storage, which is convenient for users, especially in low‐resource settings [ 59 ]. The first RPA assay was developed to detect TB targeting putative insertion sequence regions of MTB, IS6110 and IS1081. The RPA reactions were incubated at 39°C using an integrated heating and fluorescence detection machine (Twista, TwistDx) for 20 min with detection limits of 6.25 fg and 20 fg for IS6110 and IS1081, respectively, when testing purified MTB genomic DNA [ 24 ]. Furthermore, when testing with sputum and respiratory specimens from suspected TB patients, the sensitivity and specificity of RPA were 87.5% and 95.4% for IS6110 and 91.4% and 100% for IS1081 compared to the culture identification method. The end‐point detection of IS6110 and IS1081 RPA products was recently demonstrated through lateral flow and SYBR Green I assays [ 25 , 26 ]. RPA assay targeting IS1081 combined with lateral flow (LF‐RPA) provides a rapid and visual detection method for MTB. The system could work well at a broad range of temperatures, 25°C–45°C, and reach a detectable level even within 5 min that could detect as few as 5 genome copies of MTB DNA with no cross‐reaction with other bacteria. LF‐RPA exhibited 100% sensitivity and 97.92% specificity compared to the culture [ 25 ]. The RPA reaction coupled with a simple DNA‐intercalating dye, SYBR Green I, was demonstrated to detect MTB DNA [ 26 ]. Due to the simple colourimetric signal detection property of SYBR Green I, the platform offers an intermediate outcome in only 15–20 min with no additional specialized instruments, permitting the possibility of developing a fully portable POCT device [ 26 ]. All studies targeting IS1081 showed greater MTB detection sensitivity than studies targeting IS6110, possibly because IS1081 is present in all MTB strains, whereas IS6110 RPA may be at a disadvantage in IS6110‐lacking MTB strains, as previously described. When combined with fluorescent probe assays to establish a rapid and sensitive assay, RPA could detect novel specific targets for MTB, including the mts90 and Ag85B (antigen 85B) genes [ 27 , 28 ]. mts90 is specific to M. tuberculosis with no cross‐reaction to other MTB species, such as M. bovis [ 27 ], while Ag85B has been previously developed for the application of TB diagnosis [ 62 ]. This real‐time RPA‐based novel target provides a promising approach for rapidly diagnosing TB. RPA‐based TB assays could be integrated into POCT in low‐resource settings due to their rapidity, flexibility, cold chain‐independent transportation, and low energy consumption compared to other amplification technologies. However, the FDA and CE have not approved RPA assays to date. RPA commercial kits are estimated to be expensive compared to other isothermal amplification platforms, such as LAMP. Furthermore, some weaknesses of RPA may be considered when developing the assay. The presence of high‐molecular‐weight proteins impedes the verification of RPA amplification by gel electrophoresis. Thus, a postamplification purification step is needed. Additionally, the conditions of the RPA reaction are rigorous, leading to the difficulty of detection using crude samples [ 47 , 59 ]. 2.5. Rolling Cycle Amplification (RCA) RCA is an isothermal DNA amplification method adapted from the in vivo RCA mechanism and was introduced in the mid‐1990s. The reaction has circular DNA as the primary template type, which presents a restriction for some purposes. However, RCA is a simple and efficient isothermal enzymatic process that utilises unique DNA polymerase with strand displacement activity (such as Phi29 DNA polymerase) [ 63 ]. RCA is operated under a constant temperature between 30°C and 37°C and produces approximately 10 3 copies of the circular template within an hour. The RCA amplicon can be detected through various signal readout techniques (Table 1 ), of which gel electrophoresis is the most common. Additionally, incorporating fluorescent dyes into RCA products via fluorophore‐conjugated dNTPs during RCA or fluorophore‐tethered complementary strand hybridisation permits easy visualisation using fluorescence spectroscopy or microscopy. Nanoparticles (such as gold nanoparticles) or magnetic beads can be assembled onto RCA products via complementary oligonucleotides to visualise RCA products [ 63 ]. Even though the RCA assay has been in development for almost three decades, no RCA‐based diagnostic kits have yet been commercially launched [ 60 ]. The development of RCA for MTB detection is limited compared to that of other well‐known isothermal amplification techniques, such as LAMP, SDA, HDA, and RPA. The RCA‐based assay for MTB detection was first revealed in 2011, in which the RCA reaction targeting the IS6110 sequence was performed on the bead. After ligation and amplification, the obtained ssDNA product was monitored by fluorescent probes, giving a detection limit of 10,000 CFU/mL, close to the sensitivity required for clinical diagnosis [ 29 ]. The ultrasensitive method for MTB‐specific DNA detection was performed by dark‐field microscopic imaging with a gold nanoparticle counting strategy and RCA. As little as 10 fM MTB DNA targets can still be differentiated from the blank, and there was no cross‐reactivity with the genomic DNA derived from humans and bacteria causing lung disease [ 30 ]. The success of using RCA to detect MTB in a direct clinical sample has been demonstrated. Hyperbranched RCA (HRCA) with a padlock probe designed by the IS6110 sequence was developed and evaluated for efficacy in directly detecting MTB from 136 sputum specimens from TB patients [ 31 ]. The detection sensitivity was 77.2%, which was slightly lower than that of real‐time PCR (79.4%) and culture (80.9%). However, the specificity of HRCA (98.6%) was similar to that of real‐time PCR and smear/culture methods (100%). Under the optimal conditions, HRCA showed high sensitivity in detecting culture suspensions as low as 200 CFU/mL of the MTB H37Rv reference strain, which is close to the gold‐standard culture method for identifying MTB (10 to 100 CFU/mL). A smartphone‐assisted fluorescent analysis combined with fluorescent copper nanoprobes in a specific amplification has been proven as a new detection platform for diagnosing TB. PCR, padlock probe ligation, and RCA were combined to amplify the IS6110 targets in TB samples. The results were then visualised through excitation from a UV transilluminator and quantified with a smartphone. After all conditions were optimised, this sensing platform had a detection limit of 5 fg/μL for the IS6110 sequence of MTB dsDNA [ 32 ]. 3. Conclusions The significant progress achieved in the field of isothermal amplification techniques has sparked a new era in the detection of TB. Its simplicity (operated at a single temperature using only a simple heating box), rapidity (average less than 1 h), robustness (low detection limit with no cross‐reactivity), and affordable price make it a powerful candidate for improving the early detection and management of TB, particularly in resource‐constrained environments where infrastructure and access to advanced laboratory equipment are limited. Several putative isothermal amplification techniques have been developed; however, only a few are notable in the field of TB diagnosis. The WHO‐endorsed LAMP assay offers the advantages of high sensitivity and excellent performance in actual clinical samples. Many health care systems have widely adopted LAMP as a substitute for sputum smear microscopy in the detection of TB. LAMP has scaled up TB testing and improved access to diagnostic tools, particularly in regions with a high burden of TB and limited health care infrastructure. During the last decade, RPA has garnered much attention as a rapid tool to detect TB. The RPA‐based method demonstrates promising potential for TB detection and drug‐susceptibility testing due to its rapidity, simplicity, and sensitivity. However, RPA reagents can be relatively expensive, especially compared to LAMP. Furthermore, ensuring the accuracy and reliability of RPA‐based TB tests still requires rigorous validation. Other isothermal amplification techniques discussed in this review may exhibit good performance for TB detection. Several of them are still in the study phase and have the potential for further development. A summary of the advantages, clinical limitations, and cost considerations associated with isothermal amplification techniques for TB diagnosis is provided in Table 2 of this review. TABLE 2 Summary of the advantages, clinical limitations, and cost considerations of common isothermal amplification techniques used to detect TB. Amplification techniques Advantages Clinical limitations Cost a LAMP – WHO endorsement – Validated through numerous clinical studies – Resistant to PCR inhibitors – Cost‐effective (approximately $6 per test, as targeted by WHO) – Commercial availability (e.g., Loopamp) – Complex primer design – Highly prone to carryover contamination – Often relies on indirect detection (e.g., turbidity or nonspecific dyes), resulting in a false‐positive reaction – Limited suitability for drug resistance detection $6/1 reactions (Loopamp MTBC detection Kit, Eiken Chemical Co. Ltd.) SDA – Multiplexing Capability – Automated and high‐throughput options available (e.g., BDProbeTec ET) – Requires initial heat denaturation – Long reaction time (2–2.5 h) – Costly BDProbeTec ET detection equipment – Limited clinical validation $1973/96 reactions (GenomiPhi HY Ready‐To‐Go DNA Amplification Kit, Cytiva) HDA – Compatibility with multiple detection platforms – Available with POCT integration (TINY system) – Relatively low sensitivity – Require additional primer modification to reduce false‐positive – Limited clinical validation $592/50 reactions (IsoAmpII Universal tHDA kit, New England Biolabs Inc.) RPA – Fastest amplification system – Multiplexing capability – Compatibility with multiple detection platforms – Resistant to PCR inhibitors – Cold Chain Independent – Regulatory approval is lacking, limiting clinical use – Licensing is restricted (commercialised by TwistDx Limited) £372/96 reactions (TwistAmp Basic, TwistDx Limited.) RCA – High amplification fidelity – Compatibility with multiple detection platforms – High sensitivity in advanced formats (e.g., hyperbranched RCA) – Limited clinical validation – Less compatible with high‐throughput platforms – Requires multiple steps, limiting POCT suitability – Limited to circular DNA templates $343/100 reactions (phi29‐XT RCA DNA Amplification Kits, New England Biolabs Inc.) a Cost data were obtained from the respective manufacturers' product information. The future direction of isothermal amplification for TB detection is clearly centred around LAMP, which has established itself as the leading technique due to its robust performance, widespread adoption, and endorsement by the WHO. Its dominance is reflected in the consistently high number of related publications and extensive validation in the field. While older techniques like SDA have declined and essentially phased out following LAMP's emergence, newer techniques such as RPA are gaining interest. RPA offers key advantages, including rapid amplification with high sensitivity and specificity, as demonstrated in validation studies; however, its broader application is limited by licensing restrictions. In the case of RCA, further clinical validation is still needed, alongside overcoming challenges related to protocol complexity. HDA shows limited impact, lacks sufficient validation, and falls short of current research trends. Emerging other isothermal techniques, such as PSR, CPA, and MCDA, show promise with a rising publication trend, indicating ongoing innovation and potential for future breakthroughs. However, until these methods undergo rigorous validation and field testing, LAMP will likely remain the mainstay for TB diagnosis among isothermal amplification techniques. Selecting the appropriate target for detecting TB is crucial for the accuracy and sensitivity of the test. Several common targets, for example, 16S rRNA, hsp , gyrB , rpoB , IS6110, and IS1081, have been used in isothermal amplification of the TB assay. The choice of target depends on the specific goals of the test, such as diagnosis, drug susceptibility testing, or genotyping for epidemiological studies. Additionally, the selection of targets should consider factors including sensitivity and specificity. A combination of targets may be used to improve accuracy and reduce the risk of false positives or negatives. Various platforms can be employed to detect the amplification products generated by these isothermal techniques, such as visual detection (turbidity or fluorescent dye), portable fluorescence readers, electrochemical detection, and lateral flow assays. The choice of detection platform depends on the specific isothermal amplification technique, the resources available, and the intended application. Some platforms are more suitable for POCT or field use, while others are better suited for research or clinical laboratories. Additionally, the choice may be influenced by the need for quantitative data, sensitivity and specificity requirements, and cost considerations. Isothermal amplification is a powerful and rapid molecular test that can be applied in POCT for TB detection. A TB‐POCT might be deployable at the most decentralised levels of health care and provide results promptly. These virtues may allow early treatment within a few hours and prevent patient loss to follow‐up, which follows the WHO's policy to achieve the End TB Strategy. Author Contributions N.J. reviewed and analysed the data, wrote the main manuscript, and prepared table and figure. P.R. conceptualized the manuscript, did project administration and revised the manuscript. All authors read and approved the final manuscript. Disclosure The authors have nothing to report. 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