Isothermal Nucleic Acid Amplification Technique Research Articles

Endonuclease Q as a robust enhancer for nucleic acid amplification

Isothermal nucleic acid amplification techniques are attracting increasing attention in molecular diagnosis and biotechnology. However, most existing techniques are complicated by the need for intricate primer design and numerous enzymes and primers. Here, we have developed a simple method, termed NAQ, that employs adding both endonuclease Q (EndoQ) and dUTP/dITP to conventional rolling circle amplification reactions to increase DNA amplification. NAQ does not require intricate primer design or DNA sequence-specific enzymes, and existing isothermal amplification techniques could be readily adapted to include both EndoQ and dUTP/dITP.

Evaluation of the effect of outer primer structure, and inner primer linker sequences, in the performance of Loop-mediated isothermal amplification

Loop-mediated isothermal amplification, or LAMP, is nowadays the most popular isothermal nucleic acid amplification technique. This technique implements a minimum of four primers, named outer (F3/B3) and inner primers (FIP/BIP). The inner primers hybridize in two distinct regions, and some studies have reported that the usage of a linker, typically composed of four thymines, in the middle of these primers can improve assay performance. In addition to this, dual-priming oligonucleotides, DPO, have been reported to provide highly specific reducing non-specific amplifications. Considering the large number of primers implemented in LAMP assays, in the current study the suitability of DPO primers replacing regular outer primers; and their combination with different linker sequences in the inner primers were explored. The results demonstrated that replacing standard F3/B3 by DPO primers does not significantly affect that overall performance of the assay, and provides additional stability to temperature changes. This observations were consistent regardless the type of linker implemented in the inner primers, out of which in the current study a linker composed of thymines significantly outperformed the other options tested, most likely due to a combination of sequence and physical structure.

Development and application of a dual ERA method for the detection of Feline Calicivirus and Feline Herpesvirus Type I

Feline calicivirus (FCV) and feline herpesvirus type I (FHV-1) are the most common viral pathogens responsible for cat respiratory diseases, and coinfection with these two pathogens is often found. In veterinary clinics, the main diagnostic methods for FCV and FHV-1 are test strips and polymerase chain reaction (PCR). However, the sensitivity of test strips are not sufficient, and PCR is time-consuming. Therefore, developing a rapid and high-performance clinical diagnostic test is imperative for the prevention and treatment of these diseases. Enzymatic recombinase amplification (ERA) is an automated isothermal nucleic acid amplification technique that maintains a constant temperature, and is both rapid and highly accurate. In this study, a dual ERA method was developed using the Exo probe for a differential detection of FCV and FHV-1. This dual ERA method demonstrated high performance with the detection limit of 101 copies for both viruses, and no cross-reactions with feline parvovirus virus and F81 cells. To test the utility of the method for clinical applications, 50 nasopharyngeal swabs from cats with respiratory symptoms were collected and tested. The positive rates of FCV and FHV-1 were 40% (20/50, 95% confidence interval [CI], 26.4 to 54.8%) and 14% (7/50, 95% CI, 5.8 to 26.7%), respectively. The rate of coinfection with FCV and FHV-1 was 10% (5/50, 95% CI, 3.3 to 21.8%). These results were in agreement with those found using quantitative real-time PCR. Therefore, this dual ERA method is a novel and efficient clinical diagnostic tool for FCV and FHV-1 detection.

AutoPLP: A Padlock Probe Design Pipeline for Zoonotic Pathogens.

Emergence of novel zoonotic infections among the human population has increased the burden on global healthcare systems to curb their spread. To meet the evolutionary agility of pathogens, it is essential to revamp the existing diagnostic methods for early detection and characterization of the pathogens at the molecular level. Padlock probes (PLPs), which can leverage the power of isothermal nucleic acid amplification techniques (NAAT) such as rolling circle amplification (RCA), are known for their high sensitivity and specificity in detecting a diverse pathogen panel of interest. However, due to the complexity involved in deciding the target regions for PLP design and the need for optimization of multiple experimental parameters, the applicability of RCA has been limited in point-of-care testing for pathogen detection. To address this gap, we have developed a novel and integrated PLP design pipeline named AutoPLP, which can automate the probe design process for a diverse pathogen panel of interest. The pipeline is composed of three modules which can perform sequence data curation, multiple sequence alignment, conservation analysis, filtration based on experimental parameters (Tm, GC content, and secondary structure formation), and in silico probe validation via potential cross-hybridization check with host genome. The modules can also take into account the backbone and restriction site information, appropriate combinations of which are incorporated along with the probe arms to design a complete probe sequence. The potential applications of AutoPLP are showcased through the design of PLPs for the detection of rabies virus and drug-resistant strains of Mycobacterium tuberculosis.

Rapid detection of bovine rotavirus a by isothermal reverse transcription recombinase polymerase amplification assays

BackgroundBovine rotavirus A (BRVA) is considered to be the most common pathogen of severe diarrhea in cattle worldwide, which could lead to the death of newborn calves and cause the significant economic losses to the cattle industry. As a novel isothermal nucleic acid amplification technique, recombinase polymerase amplification (RPA) has been applied widely for the rapid detection of different important pathogens in human and animals.ResultsAn RT-RPA assay based on the real time fluorescence monitoring (real-time RT-RPA) and an RT-RPA assay combined with a lateral flow strip (LFS RT-RPA) were successfully developed by targeting the VP6 gene of BRVA. The RT-RPA assays allowed the exponential amplification of the target fragment in 20 min. After incubation of the LFS RT-RPA on a metal bath at 40 °C, the results were displayed on the lateral flow strip within 5 min, while real-time RT-RPA allowed the real-time observation of the results in Genie III at 42 °C. Both of the two assays showed high specificity for BRVA without any cross-reaction with the other tested pathogens causing diarrhea in cattle. With the standard RNA of BRVA serving as a template, the limit of detection for real-time RT-RPA and LFS RT-RPA were 1.4 × 102 copies per reaction and 1.4 × 101 copies per reaction, respectively. In the 134 fecal samples collected from cattle with diarrhea, the BRVA positive rate were 45.52% (61/134) and 46.27% (62/134) in real-time RT-RPA and LFS RT-RPA, respectively. Compared to a previously published real-time PCR, the real-time RT-RPA and LFS RT-RPA showed a diagnostic specificity of 100%, diagnostic sensitivity of 98.39% and 100%, and a kappa coefficient of 0.985 and 1.0, respectively.ConclusionsIn this study, BRVA was successfully detected in cattle fecal samples by the developed real-time RT-RPA and LFS RT-RPA assays. The developed RT-RPA assays had great potential for the rapid detection of BRVA in under-equipped diagnostic laboratory and the point-of-need diagnosis at quarantine stations and farms, which is of great importance to control BRVA-associated diarrhea in cattle herds.

Rapid and simple detection of influenza virus via isothermal amplification lateral flow assay

Respiratory illness caused by influenza virus is a serious public health problem worldwide. As the symptoms of influenza virus infection are similar to those of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, it is essential to distinguish these two viruses. Therefore, to properly respond to a pathogen, a detection method that is capable of rapid and accurate diagnosis in a hospital or at home is required. To satisfy this need, we applied loop-mediated isothermal amplification (LAMP) and an isothermal nucleic acid amplification technique, along with a system to analyze the results without specialized equipment, a lateral flow assay (LFA). Using the platform developed in this study, all processes, from sample preparation to detection, can be performed without special equipment. Unlike existing PCR methods, the nucleic acid amplification can be performed in the field because hot packs do not require electricity. Thus, the designed platform can provide rapid results without the need to transport the samples to a laboratory or hospital. These advantages are not limited to operations in developing countries with poor access to medical systems. In conclusion, the developed technology is a promising tool for infectious disease management that allows for rapid identification of infectious diseases and appropriate treatment of patients.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-022-04090-8.

Evaluation of electropolymerized molecularly imprinted polymers (E-MIPs) on disposable electrodes for detection of SARS-CoV-2 in saliva

We investigate electropolymerized molecularly imprinted polymers (E-MIPs) for the selective recognition of SARS-CoV-2 whole virus. E-MIPs imprinted with SARS-CoV-2 pseudoparticles (pps) were electrochemically deposited onto screen printed electrodes by reductive electropolymerization, using the water-soluble N-hydroxmethylacrylamide (NHMA) as functional monomer and crosslinked with N,N′-methylenebisacrylamide (MBAm). E-MIPs for SARS-CoV-2 showed selectivity for template SARS-CoV-2 pps, with an imprinting factor of 3:1, and specificity (significance = 0.06) when cross-reacted with other respiratory viruses. E-MIPs detected the presence of SARS-CoV-2 pps in <10 min with a limit of detection of 4.9 log10 pfu/mL, suggesting their suitability for detection of SARS-CoV-2 with minimal sample preparation. Using electrochemical impedance spectroscopy (EIS) and principal component analysis (PCA), the capture of SARS-CoV-2 from real patient saliva samples was also evaluated. Fifteen confirmed COVID-19 positive and nine COVID-19 negative saliva samples were compared against the established loop-mediated isothermal nucleic acid amplification (LAMP) technique used by the UK National Health Service. EIS data demonstrated a PCA discrimination between positive and negative LAMP samples. A threshold real impedance signal (ZRe) ≫ 4000 Ω and a corresponding charge transfer resistance (RCT) ≫ 6000 Ω was indicative of absence of virus (COVID-19 negative) in agreement with values obtained for our control non-imprinted polymer control. A ZRe at or below a threshold value of 600 Ω with a corresponding RCT of <1200 Ω was indicative of a COVID-19 positive sample. The presence of virus was confirmed by treatment of E-MIPs with a SARS-CoV-2 specific monoclonal antibody.

Ultrasensitive Uracil-DNA Glycosylase Activity Assay and Its Inhibitor Screening Based on Primer Remodeling Jointly via Repair Enzyme and Polymerase.

The development of isothermal nucleic acid amplification techniques has great significance for highly sensitive biosensing in modern biology and biomedicine. A facile and robust exponential rolling circle amplification (RCA) strategy is proposed based on primer-remodeling amplification jointly via a repair enzyme and polymerase, and uracil-DNA glycosylase (UDG) is selected as a model analyte. Two kinds of complexes, complex I and complex II, are preprepared by hybridizing a circular template (CT) with a uracil-containing hairpin probe and tetrahydrofuran abasic site mimic (AP site)-embedded fluorescence-quenched probe (AFP), respectively. The target UDG specifically binds to complex I, resulting in the generation of an AP site, followed by cleavage via endonuclease IV (Endo IV) and the successive trimming of unmatched 3' terminus via phi29 DNA polymerase, thus producing a useable primer-CT complex that actuates the primary RCA. Then, numerous complex II anneal with the first-generation RCA product (RP), generating a complex II-RP assembly containing AP sites within the DNA duplex. With the aid of Endo IV and phi29, AFP, as a pre-primer in complex II, is converted into a mature primer to initiate additional rounds of RCA. So, countless AFPs are cleaved, releasing remarkably strong fluorescent signals. The biosensor is demonstrated to enable rapid and accurate detection of the UDG activity with an improved detection limit as low as 4.7 × 10-5 U·mL-1. Moreover, this biosensor is successfully applied for UDG inhibitor screening and complicated biological samples analysis. Compared to the previous exponential RCA methods, our proposed strategy offers additional advantages, including excellent stability, optional design of CT, and simplified operating steps. Therefore, this proposed strategy may create a useful and practical platform for ultrasensitive detection of low levels of analytes in clinical diagnosis and fundamental biomedicine research.

Naked-eye detection strategies coupled with isothermal nucleic acid amplification techniques for the detection of human pathogens.

Nucleic acid amplification-based techniques have gained acceptance by the scientific, and general, community as reference methodologies for many different applications. Since the development of the gold standard of these techniques, polymerase chain reaction (PCR), back in the 1980s many improvements have been made, and alternative techniques emerged reporting improvements over PCR. Among these, isothermal amplification approaches resulted of particular interest as could overcome the need of specialized equipment to accurately control temperature changes, but it was after year 2000 that these techniques have flourished in a huge number of novel alternatives with many different degrees of complexities and requirements. An added value is their possibility to be combined with many different naked-eye detection strategies, simplifying the resources needed, allowing to reduce cost, and serving as the basis for novel developments of lab-on-chip systems, and miniaturized devices, for point-of-care testing. In this review, we will go over different types of naked-eye detection strategies, combined with isothermal amplification. This will provide the readers up-to-date information for them to select the most appropriate strategies depending on the particular needs and resources for their experimental setup.

Multiple Cross Displacement Amplification Coupled with Lateral Flow Biosensor (MCDA-LFB) for rapid detection of Legionella pneumophila

BackgroundLegionella pneumophila is an opportunistic waterborne pathogen of significant public health problems, which can cause serious human respiratory diseases (Legionnaires’ disease). Multiple cross displacement amplification (MCDA), a isothermal nucleic acid amplification technique, has been applied in the rapid detection of several bacterial agents. In this report, we developed a MCDA coupled with Nanoparticles-based Lateral Flow Biosensor (MCDA-LFB) for the rapid detection of L. pneumophila.ResultsA set of 10 primers based on the L. pneumophila specific mip gene to specifically identify 10 different target sequence regions of L. pneumophila was designed. The optimal time and temperature for amplification are 57 min and 65 °C. The limit of detection (LoD) is 10 fg in pure cultures of L. pneumophila. No cross-reaction was obtained and the specificity of MCDA-LFB assay was 100%. The whole process of the assay, including 20 min of DNA preparation, 35 min of L. pneumophila-MCDA reaction, and 2 min of sensor strip reaction, took a total of 57 min (less than 1 h). Among 88 specimens for clinical evaluation, 5 (5.68%) samples were L. pneumophila-positive by MCDA-LFB and traditional culture method, while 4(4.55%) samples were L. pneumophila-positive by PCR method targeting mip gene. Compared with culture method, the diagnostic accuracy of MCDA-LFB method was higher.ConclusionsIn summary, the L. pneumophila-MCDA-LFB method we successfully developed is a simple, fast, reliable and sensitive diagnostic tool, which can be widely used in basic and clinical laboratories.

Auto-cycling primer extension for amplified microRNA detection.

The current polymerase-based nucleic acid amplification techniques cannot maintain continuous polymerization reactions unless by changing the temperature or adding additional reagents (e.g. the second enzyme or betaine), which undoubtedly increases the cost and operation steps. Herein, a new isothermal nucleic acid amplification technique, termed auto-cycling primer extension (APE), is presented. It repeatedly extends short DNA primers to longer DNA hairpins, by combining a DNA-based copy-and-release hairpin (CRH) and palindromic sequence design. The experimental results showed that we could realize the amplification detection of miRNA by a reasonable probe design.

Current and innovative methods for the diagnosis of COVID‑19 infection (Review).

The Coronavirus Disease 2019 (COVID-19) pandemic has forced the scientific community to rapidly develop highly reliable diagnostic methods in order to effectively and accurately diagnose this pathology, thus limiting the spread of infection. Although the structural and molecular characteristics of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were initially unknown, various diagnostic strategies useful for making a correct diagnosis of COVID-19 have been rapidly developed by private research laboratories and biomedical companies. At present, rapid antigen or antibody tests, immunoenzymatic serological tests and molecular tests based on RT-PCR are the most widely used and validated techniques worldwide. Apart from these conventional methods, other techniques, including isothermal nucleic acid amplification techniques, clusters of regularly inter-spaced short palindromic repeats/Cas (CRISPR/Cas)-based approaches or digital PCR methods are currently used in research contexts or are awaiting approval for diagnostic use by competent authorities. In order to provide guidance for the correct use of COVID-19 diagnostic tests, the present review describes the diagnostic strategies available which may be used for the diagnosis of COVID-19 infection in both clinical and research settings. In particular, the technical and instrumental characteristics of the diagnostic methods used are described herein. In addition, updated and detailed information about the type of sample, the modality and the timing of use of specific tests are also discussed.

LAMP in Neglected Tropical Diseases: A Focus on Parasites.

Neglected Tropical Diseases (NTDs), particularly those caused by parasites, remain a major Public Health problem in tropical and subtropical regions, with 10% of the world population being infected. Their management and control have been traditionally hampered, among other factors, by the difficulty to deploy rapid, specific, and affordable diagnostic tools in low resource settings. This is especially true for complex PCR-based methods. Isothermal nucleic acid amplification techniques, particularly loop-mediated isothermal amplification (LAMP), appeared in the early 21st century as an alternative to PCR, allowing for a much more affordable molecular diagnostic. Here, we present the status of LAMP assays development in parasite-caused NTDs. We address the progress made in different research applications of the technique: xenomonitoring, epidemiological studies, work in animal models and clinical application both for diagnosis and evaluation of treatment success. Finally, we try to shed a light on the improvements needed to achieve a true point-of-care test and the future perspectives in this field.

A Multi-Country, Single-Blinded, Phase 2 Study to Evaluate a Point-of-Need System for Rapid Detection of Leishmaniasis and Its Implementation in Endemic Settings.

With the advancement of isothermal nucleic acid amplification techniques, detection of the pathogenic DNA in clinical samples at point-of-need is no longer a dream. The newly developed recombinase polymerase amplification (RPA) assay incorporated in a suitcase laboratory has shown promising diagnostic efficacy over real-time PCR in detection of leishmania DNA from clinical samples. For broader application of this point-of-need system, we undertook a current multi-country diagnostic evaluation study towards establishing this technique in different endemic settings which would be beneficial for the ongoing elimination programs for leishmaniasis. For this study purpose, clinical samples from confirmed visceral leishmaniasis (VL) and post-kala-azar dermal leishmaniasis (PKDL) patients were subjected to both real-time PCR and RPA assay in Bangladesh, India, and Nepal. Further skin samples from confirmed cutaneous leishmaniasis (CL) patients were also included from Sri Lanka. A total of 450 clinical samples from VL patients, 429 from PKDL patients, 47 from CL patients, and 322 from endemic healthy/healthy controls were under investigation to determine the diagnostic efficacy of RPA assay in comparison to real-time PCR. A comparative sensitivity of both methods was found where real-time PCR and RPA assay showed 96.86% (95% CI: 94.45–98.42) and 88.85% (95% CI: 85.08–91.96) sensitivity respectively in the diagnosis of VL cases. This new isothermal method also exhibited promising diagnostic sensitivity (93.50%) for PKDL cases, when a skin sample was used. Due to variation in the sequence of target amplicons, RPA assay showed comparatively lower sensitivity (55.32%) than that of real-time PCR in Sri Lanka for the diagnosis of CL cases. Except for India, the assay presented absolute specificity in the rest of the sites. Excellent concordance between the two molecular methods towards detection of leishmania DNA in clinical samples substantiates the application of RPA assay incorporated in a suitcase laboratory for point-of-need diagnosis of VL and PKDL in low resource endemic settings. However, further improvisation of the method is necessary for diagnosis of CL.

A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions

Isothermal nucleic acid amplification techniques are used to detect a variety of molecules such as RNAs, DNAs and proteins. When compared to the gold standard polymerase chain reaction, isothermal methods are rapid and simple but often have lower specificity and sensitivity. In this review, we provide a summary of reaction additives and techniques that enhance the sensitivity, specificity, stability, robustness, or speed of isothermal nucleic acid amplification reactions used for sensing applications. We also provide the mechanism and purpose of each enhancement technique, which can provide guidance when selecting reaction additives to optimize novel isothermal amplification systems and assays.

Rapid genotyping of bacterial leaf blight resistant genes of rice using loop-mediated isothermal amplification assay.

The use of resistant (R) genes is the most effective strategy to manage bacterial leaf blight (BLB) disease of rice. Several attempts were made to incorporate R genes into susceptible rice cultivars using marker-assisted backcross breeding (MABB). However, MABB relies exclusively on PCR for foreground selection of R genes, which requires expensive equipment for thermo-cycling and visualization of results; hence, it is limited to sophisticated research facilities. Isothermal nucleic acid amplification techniques such as loop-mediated isothermal amplification (LAMP) assay do not require thermo-cycling during the assay. Therefore, it will be the best alternative to PCR-based genotyping. In this study, we have developed a LAMP assay for the specific and sensitive genotyping of seven BLB resistance (R) genes viz., Xa1, Xa3, Xa4, Xa7, Xa10, Xa11, and Xa21 in rice. Gene-specific primers were designed for the LAMP assay. The LAMP assay was optimized for time, temperature, and template DNA concentration. For effective detection, incubation at 60°C for 30min was optimum for all seven R genes. A DNA intercalating dye ethidium bromide and a calorimetric dye hydroxynaphthol blue was used for result visualization. Further, sensitivity assay revealed that the LAMP assay could detect R genes at 100fg of template DNA compared to 1ng and 10pg, respectively, in conventional PCR and q-PCR assays. The LAMP assay developed in this study provides a simple, specific, sensitive, robust, and cost-effective method for foreground selection of R genes in the resistance breeding programs of resource-poor laboratory.

Single-Cell Analysis of Highly Metastatic Circulating Tumor Cells by Combining a Self-Folding Induced Release Reaction with a Cell Capture Microchip.

Nondestructive analysis of the single-cell molecular phenotype of circulating tumor cells (CTCs) is of great significance to the precise diagnosis and treatment of cancer but is also a huge challenge. To address this issue, here, we develop a facile analysis system that integrates CTCs' capture and molecular phenotype analysis. An isothermal nucleic acid amplification technique named self-folding induced release reaction (sFiR), which has high-efficiency signal amplification capabilities and can run under physiological conditions, is first developed to meet the high requirements for sensitivity and nondestructivity. By combining the sFiR with immune recognition and a single cell capture microchip, the molecular phenotype analysis of a single CTC is realized. As a model, nondestructive analysis of junction plakoglobin (JUP), an overexpressed membrane protein that is closely related to the metastasis of CTCs, is successfully achieved. Results reveal that this sFiR-based analysis system can clearly distinguish the expression of JUP in different cancer cell lines and can present quantitative information on the expression of JUP. Furthermore, the captured and analyzed CTCs maintain their basic physiological activity and can be used for drug sensitivity testing. Considering the excellent performance and ease of operation of the system, it can provide technical support for CTC-based cancer liquid biopsy and drug development.

Combinations of PCR and Isothermal Amplification Techniques Are Suitable for Fast and Sensitive Detection of SARS-CoV-2 Viral RNA.

The newly identified coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes coronavirus disease 2019 (COVID-19) and has affected over 25 million people worldwide as of August 31, 2020. To aid in the development of diagnostic kits for rapid and sensitive detection of the virus, we evaluated a combination of polymerase chain reaction (PCR) and isothermal nucleic acid amplification techniques. Here, we compared conventional PCR and loop-mediated isothermal amplification (LAMP) methods with hybrid techniques such as polymerase chain displacement reaction (PCDR) and a newly developed PCR-LAMP method. We found that the hybrid methods demonstrated higher sensitivity and assay reaction rates than those of the classic LAMP and PCR techniques and can be used to for SARS-CoV-2 detection. The proposed methods based on the modern hybrid amplification techniques markedly improve virus detection and, therefore, can be extremely useful in the development of new diagnostic kits.

Multiplex Recombinase Polymerase Amplification Assay for Simultaneous Detection of Treponema pallidum and Haemophilus ducreyi in Yaws-Like Lesions.

Yaws is a skin debilitating disease caused by Treponema pallidum subspecies pertenue with most cases reported in children. World Health Organization (WHO) aims at total eradication of this disease through mass treatment of suspected cases followed by an intensive follow-up program. However, effective diagnosis is pivotal in the successful implementation of this control program. Recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification technique offers a wider range of differentiation of pathogens including those isolated from chronic skin ulcers with similar characteristics such as Haemophilus ducreyi (H. ducreyi). We have developed a RPA assay for the simultaneous detection of Treponema pallidum (T. pallidum) and H. ducreyi (TPHD-RPA). The assay demonstrated no cross-reaction with other pathogens and enable detection of T. pallidum and H. ducreyi within 15 min at 42 °C. The RPA assay was validated with 49 clinical samples from individuals confirmed to have yaws by serological tests. Comparing the developed assay with commercial multiplex real-time PCR, the assay demonstrated 94% and 95% sensitivity for T. pallidum and H. ducreyi, respectively and 100% specificity. This simple novel TPHD-RPA assay enables the rapid detection of both T. pallidum and H. ducreyi in yaws-like lesions. This test could support the yaws eradication efforts by ensuring reliable diagnosis, to enable monitoring of program success and planning of follow-up interventions at the community level.

Isothermal nucleic acid amplification techniques for detection and identification of pathogenic fungi: A review.

Fungal infections have increased during the last years due to the AIDS epidemic and immunosuppressive therapies. The available diagnostic methods, such as culture, histopathology and serology, have several drawbacks regarding sensitivity, specificity and time-consuming, while molecular methods are still expensive and dependent on many devices. In order to overcome these challenges, isothermal nucleic acid amplification techniques (INAT) arose as promising diagnostic methods for infectious diseases. This review aimed to present and discuss the main contributions of the isothermal nucleic acid amplification techniques applied in medical mycology. Papers containing terms for each INAT (NASBA, RCA, LAMP, CPA, SDA, HAD or PSR) and the terms 'mycoses' or 'disease, fungal' were obtained from National Center for Biotechnology Information database until August 2019. NASBA, RCA, LAMP and PSR are the INAT reported in the literature for detection and identification of pathogenic fungi. Despite the need of a previous conventional PCR, the RCA technique might also be used for genotyping or cryptic species differentiation, which may be important for the treatment of certain mycoses; nevertheless, LAMP is the most used INAT for pathogen detection. Among all INATs herein reviewed, LAMP seems to be the most appropriate method for fungal detection, since it is affordable, sensitive, specific, user-friendly, rapid, robust, equipment-free and deliverable to end-users, fulfilling all ASSURED criteria of the World Health Organization for an ideal diagnostic method.