Isothermal nucleic acid amplification and its uses in modern diagnostic technologies

Srivastava, Preeti Wanti; Prasad, Dinesh

doi:10.1007/s13205-023-03628-6

Cited by 37 publications

(13 citation statements)

References 161 publications

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“…Despite the advantages of LAMP due to its low operating cost [ 32 ], two significant limitations impede its wider application: (1) the complex 4- or 6-primer system limits the design of multiple target detection schemes; (2) false positives reduce the reliability of testing results due to the high concentration of primers and the complexity of amplicons. Compared with other isothermal amplification technologies, RPA has a fairly simple primer design and can be performed near ambient temperature (37–42 °C).…”

Section: Discussionmentioning

confidence: 99%

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection

Gong,

Zhang,

et al. 2024

Microorganisms

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Salmonella enterica serovar Indiana (S. Indiana) is among the most prevalent serovars of Salmonella and is closely associated with foodborne diseases worldwide. In this study, we combined a recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein Cas12b (CRISPR/Cas12b)-based biosensing in a one-pot platform to develop a novel one-step identification method for S. Indiana infection diagnosis. The entire RPA-CRISPR/Cas12b reaction can be completed at 41 °C within 1 h without the need for specific instruments. The optimal concentrations of Cas12b and single-guide RNA (sgRNA) for the reaction were the same at 250 nM. The single-stranded DNA (ssDNA) reporter 8C-FQ (5′-/6-FAM/CCCCCCCC/BHQ1/-3′) presented the best performance in the reaction compared with the other reporters. The limit of detection (LoD) of the RPA-CRISPR/Cas12b assay was 14.4 copies per reaction. As for specificity, we successfully identified four S. Indiana strains among twenty-two Salmonella strains without any false-positive results, presenting 100% accuracy for S. Indiana, and no cross-reactions were observed in eight other pathogens. Moreover, a total of 109 chicken carcasses were classified by the S. Indiana RPA-CRISPR assay and PCR methods from three processing points, including 43 post-shedding, 35 post-evisceration, and 31 post-chilling. There were 17 S. Indiana-positive samples identified during the whole processing step, consisting of nine post-shedding, five post-evisceration, and three post-chilling. The corresponding S. Indiana-positive rates of post-shedding, post-evisceration, and post-chilling were 20.93% (9/43), 14.29% (5/35), and 9.68% (3/31), respectively. Results from the S. Indiana one-step RPA-CRISPR/Cas12b assay were totally in agreement with those obtained using a traditional culture method, demonstrating 100% agreement with no false-positive or false-negative results observed. Altogether, the RPA-CRISPR/Cas12b assay developed in this study represents a promising, accurate, and simple diagnostic tool for S. Indiana detection.

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Section: Discussionmentioning

confidence: 99%

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection

Gong,

Zhang,

et al. 2024

Microorganisms

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“…Nucleic acid amplification technologies (NAATs) other than PCR have also been developed, the most widespread being a variety of isothermal amplification (IA) methods that do not require temperature cycling, thus providing a simpler and less costly procedure for the rapid detection of nucleic acids from clinical samples. Some IA-based tests are now incorporated into a variety of commercial molecular tests [ 23 , 31 , 32 , 33 , 34 ]. However, an important advantage of rtPCR fluorescence detection over other molecular technologies is its ability to detect microorganisms quantitatively or semi-quantitatively, based on a standard curve [ 15 ].…”

Section: New Vision That Is Changing Id Diagnosis Medical Practicementioning

confidence: 99%

Tackling Infectious Diseases with Rapid Molecular Diagnosis and Innovative Prevention

Omar,

Boissinot,

Huletsky

et al. 2024

Infectious Disease Reports

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Infectious diseases (IDs) are a leading cause of death. The diversity and adaptability of microbes represent a continuing risk to health. Combining vision with passion, our transdisciplinary medical research team has been focussing its work on the better management of infectious diseases for saving human lives over the past five decades through medical discoveries and innovations that helped change the practice of medicine. The team used a multiple-faceted and integrated approach to control infectious diseases through fundamental discoveries and by developing innovative prevention tools and rapid molecular diagnostic tests to fulfill the various unmet needs of patients and health professionals in the field of ID. In this article, as objectives, we put in context two main research areas of ID management: innovative infection prevention that is woman-controlled, and the rapid molecular diagnosis of infection and resistance. We also explain how our transdisciplinary approach encompassing specialists from diverse fields ranging from biology to engineering was instrumental in achieving success. Furthermore, we discuss our vision of the future for translational research to better tackle IDs.

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“…10 Many studies have made efforts to provide different cycling conditions to enhance the NAATs amplification step. 13 Example alternatives include the use of Loop-Mediated Isothermal Amplification (LAMP) for SARS-CoV-2 and Influenza viruses, 14 the use of Rolling Circle Amplification (RCA) for Dengue, 15 and the use of Nucleic Acid Sequence-Based Amplification (NASBA) for Zika and SARS-CoV-2. 16,17 NASBA is particularly interesting because it can operate at temperatures ranging from 37 to 42 °C, eliminating the need for specialized thermocycling equipment.…”

Section: ■ Introductionmentioning

confidence: 99%

A Single Electrochemical Biosensor Designed to Detect Any Virus

Torres-Salvador,

Ojeda,

Castro

et al. 2024

Anal. Chem.

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Viruses are the primary cause of many infectious diseases in both humans and animals. Various testing methods require an amplification step of the viral RNA sample before detection, with quantitative reverse transcription polymerase chain reaction (RT-qPCR) being one of the most widely used along with lesser-known methods like Nucleic Acid Sequence-Based Amplification (NASBA). NASBA offers several advantages, such as isothermal amplification and high selectivity for specific sequences, making it an attractive option for low-income facilities. In this research, we employed a single electrochemical biosensor (E-Biosensor) designed for potentially detecting any virus by modifying the NASBA protocol. In this modified protocol, a reverse primer is designed with an additional 22-nucleotide sequence (tag region) at the 5′-end, which is added to the NASBA process. This tag region becomes part of the final amplicon generated by NASBA. It can hybridize with a single specific E-Biosensor probe set, enabling subsequent virus detection. Using this approach, we successfully detected three different viruses with a single E-Biosensor design, demonstrating the platform's potential for virus detection.

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Isothermal nucleic acid amplification and its uses in modern diagnostic technologies

Cited by 37 publications

References 161 publications

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection

A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection

Tackling Infectious Diseases with Rapid Molecular Diagnosis and Innovative Prevention

A Single Electrochemical Biosensor Designed to Detect Any Virus

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