Diagrammatic representation of LAMP technique

Loop-mediated isothermal amplification (LAMP) is a rapid and specific alternative that can be used for point-of-care testing. Thermo Fisher Scientific has developed a novel Bst Polymerase for LAMP, offering speed, stability, and lyophilization compatibility. LAMP-based nucleic acid detection is advantageous for point-of-care diagnostics, and Thermo Fisher's improvements enhance its potential for diagnostic test developers. In this article, we will explore:


PCR in diagnostics

Polymerase chain reaction (PCR) has emerged as the dominant technique in molecular diagnostics in recent years [1]. This methodology has repeatedly demonstrated unrivalled specificity and sensitivity compared to other methods. The sensitivity of the rapid antigen test for SARS-CoV-2 was notably lower than that of RT-qPCR, especially in children younger than 5 years [2]. Likewise, digital droplet PCR demonstrated higher sensitivity and accuracy compared to FISH in several molecular abnormalities common in cancer [3].

Although PCR has the desired specificity and sensitivity, one significant setback of traditional PCR in the context of diagnostics, and more importantly at the point of care (POC)-based applications, is the time required to obtain the results. Diagnostic PCR is typically performed using a centralized testing model since the reaction requires both complex equipment required for sample prep and target amplification as well as qualified staff to conduct the diagnostic test. In POC testing, time is of the essence and rapid data generation and subsequent interpretation would be required to inform treatment options. The Molecular Biology Division of Thermo Fisher Scientific has been focused on improving isothermal amplification-based nucleic acid detection. By developing a novel polymerase with unique engineered features that are complimentary to the desired attributes of rapid and accurate molecular-based tests, Thermo Fisher Scientific is providing test developers with exceptional solutions.


Loop-mediated isothermal amplification

Simplification of nucleic acid amplification is key to removing POC challenges with PCR. To this end, isothermal-based amplification is an attractive alternative that does not require changes in temperature during the amplification process [4]. Further, these reactions can typically be performed faster than traditional PCR [4]. Thus, the application of isothermal-based amplification techniques for POC testing has the potential to circumvent at least some of the logistical challenges with PCR-based centralized testing.

Although multiple isothermal amplification techniques have been described, loop-mediated isothermal amplification (LAMP) is the most studied technique due to several inherent benefits [4]. Primarily, the LAMP reaction is extremely rapid and can produce up to 109 copies of the nucleic acid in under an hour [5]. In addition to the speed of the amplification, LAMP demonstrates exceptional specificity. “It’s one of the most specific methods compared to other isothermal methods, since most others use one primer set,” stated Urte Krukonyte, a Thermo Fisher Scientific R&D Scientist. Indeed, LAMP reactions utilize 2-3 sets of primers that can distinguish up to 8 specific locations on the template [5]. These primer sets consist of internal primers, external primers, and loop primers. The internal primers are long and complementary to 2 distinct locations while the external primers are shorter and applied at lower concentrations. In presence of template, together with the external and internal primers, the isothermal Bst DNA polymerase, which exhibits high strand displacement activity, creates a dumbbell-like DNA structure [5,6]. The speed of the reaction can be enhanced via the loop primers, that are complimentary to the sequence contained in the dumbbell-shaped DNA and thus can serve as additional starting points [6].


Enabling LAMP-based applications in point of care diagnostics

Given the benefits of LAMP, multiple infectious disease LAMP-based assays have been developed and are currently utilized [7]. Further, LAMP is gaining popularity with POC diagnostic test developers. Dr. Agne Alminaite, a Product Manager at Thermo Fisher Scientific, knows the diagnostic market demands when it comes to method development. “In diagnostics, speed is really important. For diagnostics purposes, those isothermal methods that are chosen are short, like LAMP. Which can give you result in 5-10 minutes.” However, additional improvements to LAMP-based methods are still required to improve both the performance, time to results, and the stability of LAMP-based POC diagnostic testing. “Our customers, and in general diagnostic assay developers, are using LAMP for a list of infectious agents and using those tests in remote areas,” stated Dr. Alminaite.

One such improvement is to the Bst DNA polymerase itself. Thermo Fisher Scientific sought out to develop an engineered Bst polymerase that exhibited unprecedented speed, tolerance to potential inhibitors from the sample or sample preparation method, and was suitable for developing lyophilized LAMP-based diagnostic tests. Several key attributes have been generated within this Bst Polymerase to produce the polymerase demonstrated to be exceptional. Dr. Alminaite stated, “We compared speed to at least 4-5 competitors and we can say that this is the fastest enzyme on the market. We see results in 5 minutes or less sometimes.” Further, this novel Bst polymerase has been formulated without the inclusion of glycerol. “This glycerol free buffer gives flexibility to assay developers that need to have those assays lyophilized or those who are designing assays that are based on microfluidics techniques,” Dr. Alminaite said. A lyophilized form of LAMP reactions can enhance the shelf life of the diagnostic assay developer kit as well as remove cold chain requirements for kit shipment and storage.


Summary and conclusions

LAMP-based nucleic acid detection has several advantages over conventional PCR in the context of POC diagnostic testing. Utilization of LAMP-based methods does not require sophisticated equipment or significant training to perform the test, and results can be obtained extremely quickly. Thermo Fisher Scientific has taken a great method and further improved it by developing an exceptional Bst polymerase. By introducing modifications into the polymerase, Thermo Fisher Scientific has developed the fastest Bst Polymerase on the market as well as providing diagnostic test developers the flexibility of being lyophilization compatible. Taken together, Thermo Fisher Scientific is helping diagnostic test developers realize the full potential of LAMP-based diagnostics.


References

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