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762 Results Found

  • Article
  • Open Access
6 Citations
3,343 Views
18 Pages

Protein-Based Anchoring Methods for Nucleic Acid Detection in Lateral Flow Format Assays

  • Kira Hallerbach,
  • Khadijeh Khederlou,
  • Lael Wentland,
  • Lana Senten,
  • Steven Brentano,
  • Brian Keefe and
  • Elain Fu

16 October 2023

The use of lateral flow assays to detect nucleic acid targets has many applications including point-of-care diagnostics, environmental monitoring, and food safety. A sandwich format, similar to that in protein immunoassays, is often used to capture t...

  • Review
  • Open Access
13 Citations
6,420 Views
17 Pages

Advances in Nucleic Acid Assays for Infectious Disease: The Role of Microfluidic Technology

  • Yiran Wang,
  • Jingwei Chen,
  • Zhijin Yang,
  • Xuanyu Wang,
  • Yule Zhang,
  • Mengya Chen,
  • Zizhen Ming,
  • Kaihuan Zhang,
  • Dawei Zhang and
  • Lulu Zheng

Within the fields of infectious disease diagnostics, microfluidic-based integrated technology systems have become a vital technology in enhancing the rapidity, accuracy, and portability of pathogen detection. These systems synergize microfluidic tech...

  • Article
  • Open Access
9 Citations
3,236 Views
13 Pages

Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves...

  • Article
  • Open Access
4 Citations
3,632 Views
12 Pages

Multi-Sample Preparation Assay for Isolation of Nucleic Acids Using Bio-Silica with Syringe Filters

  • Geun Su Noh,
  • Huifang Liu,
  • Myoung Gyu Kim,
  • Zhen Qiao,
  • Yoon Ok Jang and
  • Yong Shin

30 August 2020

The spin-column system for the isolation of nucleic acids (NAs) from multiple samples presents the inconvenience of repeated experimentation, time-consumption, and the risk of contamination in the process of the spin-column exchange. Herein, we propo...

  • Article
  • Open Access
11 Citations
3,234 Views
10 Pages

15 April 2022

A simple, selective, and quantitative platform for point-of-care diagnostic of COVID-19 is urgently needed as a complement in areas where resources are currently relatively scarce. To meet the needs of early diagnosis and intervention, a proof-of-con...

  • Review
  • Open Access
8 Citations
4,520 Views
14 Pages

Applied to investigate specific sequences, nucleic acid detection assays can help identify novel bacterial and viral infections. Most up-to-date systems combine isothermal amplification with Cas-mediated detection. They surpass standard PCR methods i...

  • Article
  • Open Access
5 Citations
3,439 Views
15 Pages

23 October 2023

Acinetobacter baumannii (A. baumannii) is among the main pathogens that cause nosocomial infections. The ability to rapidly and accurately detect A. baumannii and its drug resistance is essential for blocking secondary infections and guiding treatmen...

  • Article
  • Open Access
20 Citations
7,751 Views
15 Pages

24 September 2015

Bioassays based on cellulose paper substrates are gaining increasing popularity for the development of field portable and low-cost diagnostic applications. Herein, we report a paper-based nucleic acid hybridization assay using immobilized upconversi...

  • Article
  • Open Access
4 Citations
3,850 Views
14 Pages

1 December 2024

We developed a rapid and sensitive diagnostic platform that integrates isothermal viral gene amplification with a nucleic acid lateral flow assay (NALFA) to detect SARS-CoV-2 RNA. Isothermal gene amplification was performed by combining reverse trans...

  • Article
  • Open Access
7 Citations
2,476 Views
9 Pages

Comparison of the Influence of Different Nucleic Acid Extraction Assays on the Sensitivity of Trypanosoma cruzi-Specific Real-Time PCR

  • Simone Kann,
  • Wendy Zabala-Monterroza,
  • Cenia García,
  • Gustavo Concha,
  • Olfert Landt,
  • Andreas Hahn,
  • Felix Weinreich and
  • Hagen Frickmann

For the molecular diagnosis of Chagas disease by real-time PCR (polymerase chain reaction), optimization of diagnostic accuracy is desirable. The detection limit of real-time PCR assays for the diagnosis of Trypanosoma cruzi in human serum is affecte...

  • Review
  • Open Access
20 Citations
6,073 Views
24 Pages

8 November 2022

Wearable biosensors are becoming increasingly popular due to the rise in demand for non-invasive, real-time monitoring of health and personalized medicine. Traditionally, wearable biosensors have explored protein-based enzymatic and affinity-based de...

  • Review
  • Open Access
7 Citations
3,777 Views
21 Pages

16 November 2024

Gold nanoparticles (AuNPs) are highly attractive for applications in the field of biosensing, particularly for colorimetric nucleic acid detection. Their unique optical properties, which are highly sensitive to changes in their environment, make them...

  • Systematic Review
  • Open Access
5 Citations
3,714 Views
22 Pages

Current Updates on Molecular Diagnostic Assays Used for Detection of Candida auris: A Systematic Review

  • River Chun-Wai Wong,
  • Alfred Lok-Hang Lee,
  • Ingrid Yu-Ying Cheung,
  • Viola Chi-Ying Chow,
  • Margaret Ip and
  • Christopher Koon-Chi Lai

Background/Objectives: Candida auris is an emerging multidrug-resistant pathogen with the potential to cause invasive fungal infections and healthcare-associated outbreaks. Currently, there is no systematic review explicitly focusing on the up-to-dat...

  • Article
  • Open Access
14 Citations
5,130 Views
17 Pages

Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples

  • Felix Weinreich,
  • Andreas Hahn,
  • Kirsten Alexandra Eberhardt,
  • Simone Kann,
  • Thomas Köller,
  • Philipp Warnke,
  • Susann Dupke,
  • Denise Dekker,
  • Jürgen May and
  • Ulrike Loderstädt
  • + 1 author

Prior to the implementation of new diagnostic techniques, a thorough evaluation is mandatory in order to ensure diagnostic reliability. If positive samples are scarcely available, however, such evaluations can be difficult to perform. Here, we evalua...

  • Article
  • Open Access
10 Citations
4,358 Views
17 Pages

Gold–Oligonucleotide Nanoconstructs Engineered to Detect Conserved Enteroviral Nucleic Acid Sequences

  • Veeren M. Chauhan,
  • Mohamed M. Elsutohy,
  • C. Patrick McClure,
  • William L. Irving,
  • Neil Roddis and
  • Jonathan W. Aylott

14 July 2021

Enteroviruses are ubiquitous mammalian pathogens that can produce mild to life-threatening disease. We developed a multimodal, rapid, accurate and economical point-of-care biosensor that can detect nucleic acid sequences conserved amongst 96% of all...

  • Article
  • Open Access
2 Citations
3,227 Views
19 Pages

Enhancing Colorimetric Detection of Nucleic Acids on Nitrocellulose Membranes: Cutting-Edge Applications in Diagnostics and Forensics

  • Nidhi Subhashini,
  • Yannick Kerler,
  • Marcus M. Menger,
  • Olga Böhm,
  • Judith Witte,
  • Christian Stadler and
  • Alexander Griberman

5 September 2024

This study re-introduces a protein-free rapid test method for nucleic acids on paper based lateral flow assays utilizing special multichannel nitrocellulose membranes and DNA-Gold conjugates, achieving significantly enhanced sensitivity, easier proto...

  • Article
  • Open Access
7 Citations
8,822 Views
14 Pages

5 September 2012

Biopolymers are easily denatured by heating, a change in pH or chemical substances when they are immobilized on a substrate. To prevent denaturation of biopolymers, we developed a method to trap a polynucleotide on a substrate by hydrogen bonding usi...

  • Review
  • Open Access
5 Citations
2,682 Views
21 Pages

Heavy Metal Ion Detection Based on Lateral Flow Assay Technology: Principles and Applications

  • Xiaobo Xie,
  • Xinyue Hu,
  • Xin Cao,
  • Qianhui Zhou,
  • Wei Yang,
  • Ranran Yu,
  • Shuaiqi Liu,
  • Huili Hu,
  • Ji Qi and
  • Zhiyang Zhang

7 July 2025

Heavy metal ions pose a significant threat to the environment and human health due to their high toxicity and bioaccumulation. Traditional instrumentations, although sensitive, are often complex, costly, and unsuitable for on-site rapid detection of...

  • Article
  • Open Access
1 Citations
1,275 Views
16 Pages

Cobra Three-Finger Toxins Interact with RNA and DNA: Nucleic Acids as Their Putative Biological Targets

  • Alexey V. Osipov,
  • Vladislav G. Starkov,
  • Victor I. Tsetlin and
  • Yuri N. Utkin

Three-finger toxins (TFTs), including neurotoxins and cytotoxins, form one of the largest families of snake venom proteins and interact with various biological targets. Neurotoxins target proteinaceous receptors while cytotoxins interact mainly with...

  • Article
  • Open Access
35 Citations
9,778 Views
23 Pages

9 June 2011

The use of quantum dots (QDs) as donors in fluorescence resonance energy transfer (FRET) offer several advantages for the development of multiplexed solid-phase QD-FRET nucleic acid hybridization assays. Designs for multiplexing have been demonstrate...

  • Feature Paper
  • Review
  • Open Access
90 Citations
25,547 Views
24 Pages

Role of Diagnostics in Epidemiology, Management, Surveillance, and Control of Leptospirosis

  • Jane E. Sykes,
  • Krystle L. Reagan,
  • Jarlath E. Nally,
  • Renee L. Galloway and
  • David A. Haake

A One Health approach to the epidemiology, management, surveillance, and control of leptospirosis relies on accessible and accurate diagnostics that can be applied to humans and companion animals and livestock. Diagnosis should be multifaceted and ta...

  • Article
  • Open Access
10 Citations
5,962 Views
13 Pages

Microfluidic One-Pot Digital Droplet FISH Using LNA/DNA Molecular Beacons for Bacteria Detection and Absolute Quantification

  • Yu-Ting Kao,
  • Silvia Calabrese,
  • Nadine Borst,
  • Michael Lehnert,
  • Yu-Kai Lai,
  • Franziska Schlenker,
  • Peter Juelg,
  • Roland Zengerle,
  • Piotr Garstecki and
  • Felix von Stetten

12 April 2022

We demonstrate detection and quantification of bacterial load with a novel microfluidic one-pot wash-free fluorescence in situ hybridization (FISH) assay in droplets. The method offers minimal manual workload by only requiring mixing of the sample wi...

  • Perspective
  • Open Access
8 Citations
4,635 Views
13 Pages

Restriction Endonuclease-Based Assays for DNA Detection and Isothermal Exponential Signal Amplification

  • Maria Smith,
  • Kenneth Smith,
  • Alan Olstein,
  • Andrew Oleinikov and
  • Andrey Ghindilis

11 July 2020

Application of restriction endonuclease (REase) enzymes for specific detection of nucleic acids provides for high assay specificity, convenience and low cost. A direct restriction assay format is based on the specific enzymatic cleavage of a target&n...

  • Review
  • Open Access
2 Citations
4,018 Views
14 Pages

Current Analytical Methods and Challenges for the Clinical Diagnosis of Invasive Pulmonary Aspergillosis Infection

  • Madeline C. R. Schwarz,
  • Alex E. Moskaluk,
  • Joshua B. Daniels,
  • Sue VandeWoude and
  • Melissa M. Reynolds

28 November 2024

In the last decade, pulmonary fungal infections such as invasive pulmonary aspergillosis (IPA) have increased in incidence due to the increased number of immunocompromised individuals. This increase is especially problematic when considering mortalit...

  • Review
  • Open Access
22 Citations
6,907 Views
16 Pages

Technologies for Detection of Babesia microti: Advances and Challenges

  • Scott Meredith,
  • Miranda Oakley and
  • Sanjai Kumar

30 November 2021

The biology of intraerythrocytic Babesia parasites presents unique challenges for the diagnosis of human babesiosis. Antibody-based assays are highly sensitive but fail to detect early stage Babesia infections prior to seroconversion (window period)...

  • Article
  • Open Access
3 Citations
2,325 Views
15 Pages

3 March 2025

Background/Objectives: Some G-quadruplex (G4)-forming nucleic acid sequences bind a hemin cofactor to enhance its peroxidase-like activity. This has been implemented in a variety of bioanalytical assays benefiting from analyte-dependent peroxidation...

  • Article
  • Open Access
30 Citations
13,045 Views
11 Pages

18 January 2012

Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection...

  • Review
  • Open Access
8 Citations
4,558 Views
20 Pages

Fluorescence Polarization Assay for Infection Diagnostics: A Review

  • Sergei A. Eremin,
  • Liliya I. Mukhametova,
  • Vadim B. Krylov and
  • Nikolay E. Nifantiev

5 October 2024

Rapid and specific diagnosis is necessary for both the treatment and prevention of infectious diseases. Bacteria and viruses that enter the bloodstream can trigger a strong immune response in infected animals and humans. The fluorescence polarization...

  • Review
  • Open Access
10 Citations
4,568 Views
16 Pages

Personalised medicine, in which clinical management is individualised to the genotypic and phenotypic data of patients, offers a promising means by which to enhance outcomes in the management of mycobacterial pulmonary infections. In this review, we...

  • Article
  • Open Access
1,845 Views
9 Pages

26 September 2023

The detection of hepatitis E virus (HEV) RNA is the gold standard for HEV infection diagnosis. In order to address the quality control requirements for HEV RNA detection kits within China, we aimed to establish the first Chinese national standard for...

  • Review
  • Open Access
2,717 Views
24 Pages

Amplification-Free Testing of microRNA Biomarkers in Cancer

  • Bahareh Soleimanpour,
  • Juan Jose Diaz Mochon and
  • Salvatore Pernagallo

21 August 2025

Background: Circulating miRNAs have been identified as potential biomarkers for the early diagnosis and monitoring of cancers. However, limitations of polymerase chain reaction (PCR)-based methods are currently delaying the transition of miRNA resear...

  • Article
  • Open Access
76 Citations
9,148 Views
14 Pages

Electrospun Polycaprolactone Nanofibers as a Reaction Membrane for Lateral Flow Assay

  • Chee Hong Takahiro Yew,
  • Pedram Azari,
  • Jane Ru Choi,
  • Farina Muhamad and
  • Belinda Pingguan-Murphy

14 December 2018

Electrospun polycaprolactone (PCL) nanofibers have emerged as a promising material in diverse biomedical applications due to their various favorable features. However, their application in the field of biosensors such as point-of-care lateral flow as...

  • Review
  • Open Access
30 Citations
6,676 Views
29 Pages

This review is focused on the biosensing assay based on AuNPs (AuNPs) modified by proteins, peptides and nucleic acid aptamers. The unique physical properties of AuNPs allow their modification by proteins, peptides or nucleic acid aptamers by chemiso...

  • Article
  • Open Access
776 Views
21 Pages

Thermodynamic Determinants in Antibody-Free Nucleic Acid Lateral Flow Assays (AF-NALFA): Lessons from Molecular Detection of Listeria monocytogenes, Mycobacterium leprae and Leishmania amazonensis

  • Leonardo Lopes-Luz,
  • Paula Correa Neddermeyer,
  • Gabryele Cardoso Sampaio,
  • Luana Michele Alves,
  • Matheus Bernardes Torres Fogaça,
  • Djairo Pastor Saavedra,
  • Mariane Martins de Araújo Stefani and
  • Samira Bührer-Sékula

2 October 2025

Antibody-free nucleic acid lateral flow assays (AF-NALFA) are an established approach for rapid detection of amplified pathogens DNA but can yield inconsistent signals across targets. Since AF-NALFA depends on dual hybridization of probes to single-s...

  • Review
  • Open Access
7 Citations
4,203 Views
16 Pages

Sandwich Hybridization Assay for In Situ Real-Time Cyanobacterial Detection and Monitoring: A Review

  • Ping Gong,
  • Anna K. Antrim,
  • Sarah R. Bickman,
  • Emily G. Cooley and
  • Seung Ho Chung

14 August 2022

As cyanobacterial harmful algal bloom (cHAB) events increase in scale, severity, frequency, and duration around the world, rapid and accurate monitoring and characterization tools have become critically essential for regulatory and management decisio...

  • Systematic Review
  • Open Access
6 Citations
3,144 Views
14 Pages

Point-of-Care Tests for Rapid Detection of Porcine Epidemic Diarrhea Virus: A Systematic Review and Meta-Analysis

  • Renfeng Li,
  • Xiangqin Tian,
  • Junzeng Pang,
  • Linyue Li,
  • Jiakang Yuan,
  • Zhuangzhuang Tian and
  • Ziliang Wang

21 June 2022

The timely and accurate diagnosis of porcine epidemic diarrhea virus (PEDV) infection is crucial to reduce the risk of viral transmission. Therefore, the objective of this review was to evaluate the overall diagnostic accuracy of rapid point-of-care...

  • Article
  • Open Access
2 Citations
3,644 Views
11 Pages

13 December 2022

Several multiplex nucleic acid assay platforms have been developed in response to the increasing importance of nucleic acid analysis, but these assays should be optimized as per the requirements of point-of-care for clinical diagnosis. To achieve rap...

  • Article
  • Open Access
1 Citations
2,232 Views
10 Pages

MNAzyme-Assisted Nucleic Acid Lateral Flow Assay for Cost-Effective, On-Site Mercury Detection

  • Seok Hyeon Kim,
  • Yujun Kim,
  • Seokjoon Kim,
  • Eun Sung Lee,
  • Byung Seok Cha and
  • Ki Soo Park

25 September 2024

Mercury ions (Hg2+) are toxic heavy metals present in the environment that pose significant health risks. An advanced detection system could allow for a prompt response and alleviate serious damage to humans. In this study, we developed a cost-effect...

  • Article
  • Open Access
11 Citations
3,931 Views
14 Pages

15 January 2023

Zip nucleic acid (ZNA)-based genomagnetic assay was developed herein for the electrochemical detection of microRNA-34a (miR-34a), which is related to neurological disorders and cancer. The hybridization between the ZNA probe and miR-34a target was pe...

  • Article
  • Open Access
511 Views
17 Pages

Titanium Dioxide Solar Photocatalytic Microbial Inactivation Assessment Utilizing Viability Tests and a Novel Triplex qPCR Assay for Nucleic Acid Degradation Determination

  • Ioannis Paspaltsis,
  • Eirini Kanata,
  • Sotirios Sotiriadis,
  • Chrysanthi Berberidou,
  • Sophia Tsoumachidou,
  • Athanasios Arsenakis,
  • Konstantinos Xanthopoulos,
  • Dimitra Dafou,
  • Ioannis Poulios and
  • Theodoros Sklaviadis

7 November 2025

Photocatalytic oxidation of microorganisms is a powerful alternative to established disinfection approaches, applicable to a variety of water matrices. Bacterial vegetative cells, spores, fungi, and viruses, represent potential biopathogens and photo...

  • Article
  • Open Access
9 Citations
3,606 Views
10 Pages

Self-Assembly of Functional Nucleic Acid-Based Colorimetric Competition Assay for the Detection of Immunoglobulin E

  • Xuexia Lin,
  • Caiyun Yu,
  • Honggui Lin,
  • Cui Wang,
  • Jianlong Su,
  • Jie Cheng,
  • Ranjith Kumar Kankala and
  • Shu-Feng Zhou

14 May 2019

In this work, we have developed a simple and rapid colorimetric assay for the detection of immunoglobulin E (IgE) using functional nucleic acids (FNAs) and a solid-phase competition enzyme-linked immunosorbent assay (ELISA). The FNAs including aptame...

  • Article
  • Open Access
28 Citations
9,632 Views
13 Pages

4 December 2012

This paper reports a nucleic acid sandwich hybridization assay with a quantum dot (QD)-induced fluorescence resonance energy transfer (FRET) reporter system. Two label-free hemagglutinin H5 sequences (60-mer DNA and 630-nt cDNA fragment) of avian inf...

  • Feature Paper
  • Article
  • Open Access
29 Citations
6,402 Views
11 Pages

Comparative Study of Gold and Carbon Nanoparticles in Nucleic Acid Lateral Flow Assay

  • Juan Carlos Porras,
  • Mireia Bernuz,
  • Jennifer Marfa,
  • Arnau Pallares-Rusiñol,
  • Mercè Martí and
  • María Isabel Pividori

15 March 2021

A lateral flow assay (LFA) is a paper-based, point-of-need test designed to detect a specific analyte in complex samples in low-resource settings. Although LFA has been successfully used in different applications, its use is still limited when high s...

  • Article
  • Open Access
10 Citations
6,240 Views
20 Pages

Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA)

  • Anna Aviñó,
  • Carlos Cuestas-Ayllón,
  • Manuel Gutiérrez-Capitán,
  • Lluisa Vilaplana,
  • Valeria Grazu,
  • Véronique Noé,
  • Eva Balada,
  • Antonio Baldi,
  • Alex J. Félix and
  • Carlos J. Ciudad
  • + 8 authors

3 December 2022

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few...

  • Article
  • Open Access
3 Citations
3,408 Views
15 Pages

17 November 2023

This study focuses on three key aspects: (a) crude throat swab samples in a viral transport medium (VTM) as templates for RT-LAMP reactions; (b) a biotinylated DNA probe with enhanced specificity for LFA readouts; and (c) a digital semi-quantificatio...

  • Article
  • Open Access
806 Views
13 Pages

4 July 2025

Background and Objectives: The aim of this prospective study was to evaluate the diagnostic accuracy of the one-step nucleic acid amplification (OSNA) assay for the intraoperative assessment of sentinel lymph node (SN) metastases, including micrometa...

  • Review
  • Open Access
6 Citations
10,995 Views
16 Pages

Trichomoniasis is the most common non-viral sexually transmitted infection (STI) in the world. The estimated global prevalence in 2016 was 156 million adults aged 15–49. However, these data are underestimated, since the most used diagnostic met...

  • Article
  • Open Access
825 Views
17 Pages

Performance and Prognostic Relevance of Lymph Node Assessment by One-Step Nucleic Acid Amplification Assay in Rectal Cancer: A Multicenter Study

  • Qing Liu,
  • Sandra Lopez-Prades,
  • Karmele Saez de Gordoa,
  • Maite Rodrigo-Calvo,
  • Mireia Garcia,
  • Juan Ruiz Martin,
  • Angel Romo,
  • Ignacio Pinilla,
  • Jordi Tarragona and
  • Miriam Cuatrecasas
  • + 3 authors

25 June 2025

Background/Objectives: Lymph node metastases (LNM) undetected by standard hematoxylin and eosin (H&E) have been associated with unfavorable prognosis in colorectal cancer. The One-Step Nucleic Acid Amplification (OSNA) assay has demonstrated supe...

  • Review
  • Open Access
2 Citations
3,512 Views
17 Pages

Utilizing Electrochemical Biosensors as an Innovative Platform for the Rapid and On-Site Detection of Animal Viruses

  • Xun He,
  • Shan Wang,
  • Caoyuan Ma,
  • Guang-Ri Xu,
  • Jinyou Ma,
  • Hongbing Xie,
  • Wei Zhu,
  • Hongyang Liu,
  • Lei Wang and
  • Yimin Wang

8 October 2023

Animal viruses are a significant threat to animal health and are easily spread across the globe with the rise of globalization. The limitations in diagnosing and treating animal virus infections have made the transmission of diseases and animal death...

  • Article
  • Open Access
6 Citations
3,780 Views
14 Pages

Scaling Up Molecular Diagnostic Tests for Drug-Resistant Tuberculosis in Uzbekistan from 2012–2019: Are We on the Right Track?

  • Sharofiddin Yuldashev,
  • Nargiza Parpieva,
  • Salikhdjan Alimov,
  • Laziz Turaev,
  • Khasan Safaev,
  • Kostyantyn Dumchev,
  • Jamshid Gadoev,
  • Oleksandr Korotych and
  • Anthony D. Harries

Uzbekistan has a large burden of drug-resistant tuberculosis (TB). To deal with this public health threat, the National TB Program introduced rapid molecular diagnostic tests such as Xpert MTB/RIF (Xpert) and line probe assays (LPAs) for first-line a...

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