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Updated: Nov 24, 2025

Reusable Single Cell for Iterative Epigenomic Analyses

Reusable Single Cell for Iterative Epigenomic Analyses

Published on: February 11, 2022

Two stage, nested isothermal amplification in a single tube.

Mohamed El-Tholoth¹, Eman Anis, Haim H Bau

¹Department of Virology, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt. tholothvirol@mans.edu.eg.

|December 28, 2020

Summary

A new two-stage molecular test, Penn-RAMP, offers rapid, sensitive detection of animal respiratory viruses like infectious laryngotracheitis (ILT) and infectious bronchitis (IB) at the point of need. This low-cost assay requires minimal equipment and training, improving diagnostic accessibility.

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Area of Science:

Veterinary Molecular Diagnostics
Isothermal Amplification Techniques
Animal Virology

Background:

Molecular diagnostics are crucial for controlling animal respiratory diseases but conventional methods (PCR) are costly and time-consuming.
Point-of-need diagnostic tools are needed to overcome limitations of centralized laboratory testing.
Rapid and accurate diagnosis of infectious laryngotracheitis (ILT) and infectious bronchitis (IB) in poultry is economically significant.

Purpose of the Study:

To develop and validate a novel, rapid, sensitive, and point-of-need molecular diagnostic assay for animal respiratory pathogens.
To combine two isothermal amplification techniques in a single closed-tube assay for enhanced detection.
To evaluate the efficacy of the new assay for diagnosing infectious laryngotracheitis (ILT) and infectious bronchitis (IB) in chickens.

Main Methods:

Developed Penn-RAMP, a two-stage, closed-tube molecular test integrating recombinase polymerase amplification (RPA) at 38 °C and loop-mediated isothermal amplification (LAMP) at 63 °C.
Tested Penn-RAMP using clinical samples for the detection of infectious laryngotracheitis (ILT) and infectious bronchitis (IB) viruses in chickens.
Compared Penn-RAMP performance against the gold standard quantitative PCR (qPCR) assay.

Main Results:

Penn-RAMP demonstrated high sensitivity and specificity, with results concordant to qPCR.
The assay achieved a 10-fold lower limit of detection compared to both standalone LAMP and qPCR.
Penn-RAMP requires minimal instrumentation and training, operating at low temperatures (38 °C and 63 °C).

Conclusions:

Penn-RAMP is a highly sensitive, rapid, and accessible molecular diagnostic tool for animal respiratory diseases.
The integrated RPA-LAMP approach offers significant advantages over conventional PCR and standalone isothermal methods.
This technology has the potential to reduce false negatives and improve disease management in field settings.