A versatile loop-mediated isothermal amplification microchip platform for Streptococcus pneumoniae and Mycoplasma pneumoniae testing at the point of care

Hua Wang; Zhan Ma; Juanxiu Qin; Zhen Shen; Qian Liu; Xiaoyin Chen; Hengliang Wang; Zhenghua An; Wu Liu; Min Li

doi:10.1016/j.bios.2018.11.011

A versatile loop-mediated isothermal amplification microchip platform for Streptococcus pneumoniae and Mycoplasma pneumoniae testing at the point of care

Biosens Bioelectron. 2019 Feb 1:126:373-380. doi: 10.1016/j.bios.2018.11.011. Epub 2018 Nov 12.

Authors

Hua Wang¹, Zhan Ma², Juanxiu Qin³, Zhen Shen³, Qian Liu³, Xiaoyin Chen³, Hengliang Wang⁴, Zhenghua An⁴, Wu Liu⁵, Min Li⁶

Affiliations

¹ Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China. Electronic address: fdeduwh@126.com.
² Department of Laboratory Medicine, Shanghai Children's Hospital, Shanghai Jiaotong University, Shanghai, China.
³ Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China.
⁴ Dept. of Physics, Fudan University, Shanghai, China.
⁵ Department of Micro/Nano Electronics, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, China.
⁶ Department of Laboratory Medicine, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai, China. Electronic address: ruth_limin@126.com.

PMID: 30469075
DOI: 10.1016/j.bios.2018.11.011

Abstract

Community-acquired pneumonia (CAP) is the leading cause of mortality in children under five years of age, globally. Given that Streptococcus pneumoniae (S. pneumoniae) and Mycoplasma pneumoniae (M. pneumoniae) are the most common pathogens associated with CAP requiring hospital admission, a simple, low cost, highly sensitive method is in great need for immediate and early diagnosis of CAP. Herein, we report a versatile microfluidic chip platform integrated with loop-mediated isothermal amplification (LAMP) for simultaneous S. pneumoniae and M. pneumoniae testing at the point of care. The platform includes a polymer/paper microfluidic chip and a portable device. On-chip magnetic particle-based nucleic acid extraction is used for concentration of pathogens' genomic DNA and is followed by LAMP. The portable device has the function of heating the microfluidic chip, and photographing and transmitting the result to a smartphone. Complete extraction of the DNA using the microfluidic chip took ~15 min versus >1.5 h with a phenol-chloroform method. The analytical sensitivity of the assay was determined to be 20 fg by testing serial dilutions of target DNA ranging from 2 ng to 2 fg per reaction. We evaluated the clinical sensitivity and specificity of the IPµchip assay using 63 randomly selected oropharyngeal swabs and bronchoalveolar lavage fluid specimens from children. For comparison, these specimens were also tested against real-time PCR assay (M. pneumoniae), conventional PCR assay (S. pneumoniae), and culture tests (S. pneumoniae). These results yielded positive and negative predictive values for M. pneumoniae testing with the IPµchip platform of 96.9% and 100%, respectively. Compared with S. pneumoniae IPµchip, the clinical sensitivity of S. pneumoniae PCR and culture tests was 60% and 40%, respectively, while clinical specificity of the two tests was 100%. This versatile IPµchip platform has great potential for point of care testing of different kinds of pathogens, especially for developing nations.

Keywords: Loop-mediated isothermal amplification; Microfluidic chip; Pathogens; Point-of-care testing; Remote visualization.

MeSH terms

Biosensing Techniques*
Community-Acquired Infections / diagnosis
Community-Acquired Infections / microbiology
DNA, Bacterial / isolation & purification*
Humans
Microarray Analysis
Microfluidics
Mycoplasma pneumoniae / isolation & purification*
Mycoplasma pneumoniae / pathogenicity
Nucleic Acid Amplification Techniques / methods
Pneumonia / diagnosis
Pneumonia / microbiology
Point-of-Care Systems
Streptococcus pneumoniae / isolation & purification*
Streptococcus pneumoniae / pathogenicity

Substances

DNA, Bacterial