A novel linear displacement isothermal amplification with strand displacement probes (LDIA-SD) in a pocket-size device for point-of-care testing of infectious diseases

Hongchao Gou; Qijie Lin; Haiyan Shen; Kaiyuan Jia; Yucen Liang; Junhao Peng; Chunhong Zhang; Xiaoyun Qu; Yanbin Li; Jianhan Lin; Jianmin Zhang; Ming Liao

doi:10.1016/j.snb.2022.133244

A novel linear displacement isothermal amplification with strand displacement probes (LDIA-SD) in a pocket-size device for point-of-care testing of infectious diseases

Sens Actuators B Chem. 2023 Mar 15:379:133244. doi: 10.1016/j.snb.2022.133244. Epub 2022 Dec 24.

Authors

Hongchao Gou^{1

2

3

4}, Qijie Lin^{5

6

7

8

9

10}, Haiyan Shen^{1

2

3

4}, Kaiyuan Jia^{5

6

7

8

9

10}, Yucen Liang^{5

6

7

8

9

10}, Junhao Peng^{5

6

7

8

9

10}, Chunhong Zhang^{1

2

3

4}, Xiaoyun Qu^{5

6

7

8

9

10}, Yanbin Li¹¹, Jianhan Lin¹², Jianmin Zhang^{5

6

7

8

9

10}, Ming Liao^{1

5

2

3

4

6

7

8

9

10}

Affiliations

¹ Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangzhou, China.
² Guangdong Provincial Key Laboratory of Livestock Disease Prevention, Guangzhou, China.
³ Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Maoming, China.
⁴ Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Guangzhou, China.
⁵ National and Regional Joint Engineering Laboratory for Medicament of Zoonoses Prevention and Control, Guangzhou 510642, China.
⁶ Key Laboratory of Zoonoses, Ministry of Agriculture, Guangzhou 510642, China.
⁷ Key Laboratory of Zoonoses Prevention and Control of Guangdong Province, Guangzhou 510642, China.
⁸ Key Laboratory of Animal Vaccine Development, Ministry of Agriculture, Guangzhou 510642, China.
⁹ Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, China.
¹⁰ College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China.
¹¹ Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72701, USA.
¹² Key Laboratory of Agricultural Information Acquisition Technology, Ministry of Agriculture and Rural Affairs, China Agricultural University, Beijing 100083, China.

Abstract

Nucleic acid amplification is crucial for disease diagnosis, especially lethal infectious diseases such as COVID-19. Compared with PCR, isothermal amplification methods are advantageous for point-of-care testing (POCT). However, complicated primer design limits their application in detecting some short targets or sequences with abnormal GC content. Herein, we developed a novel linear displacement isothermal amplification (LDIA) method using two pairs of conventional primers and Bacillus stearothermophilus (Bst) DNA polymerase, and reactions could be accelerated by adding an extra primer. Pseudorabies virus gE (high GC content) and Salmonella fimW (low GC content) genes were used to evaluate the LDIA assay. Using strand displacement (SD) probes, a LDIA-SD method was developed to realize probe-based specific detection. Additionally, we incorporated a nucleic acid-free extraction step and a pocket-sized device to realize POCT applications of the LDIA-SD method. The LDIA-SD method has advantages including facile primer design, high sensitivity and specificity, and applicability for POCT, especially for amplification of complex sequences and detection of infectious diseases.

Keywords: Infectious diseases; Isothermal amplification; Linear displacement isothermal amplification (LDIA); Nucleic Acid Amplification; Point-of-care testing (POCT).