A LAMP-based microfluidic chip for rapid detection of pathogen in Cryptococcal meningitis

AbstractCryptococcal meningitis (CM) is a global threat with significant attributable morbidity and mortality. Information on integrated detection for CM diagnosis is still limited. This is mainly due to the presence of a large polysaccharide capsule and the tough cell wall of Cryptococcus, which makes it difficult to extract nucleic acids on the chip. In this study, we developed a LAMP-based microfluidic chip for rapid detection of pathogen in CM. We adopted 4 duplicate filtration membrane structures to improve target capture and simplify the enrichment process, and combined lyticase digestion and thermal alkaline lysisto optimize the nucleic acid extraction of Cryptococcus on the chip, and selected a portable UVA flashlight to shine the LAMP products to obtain the visual detection results which could be observed by the naked eye. This microfluidic chip, integrating sample Cryptococcus enrichment, nucleic acid extraction and LAMP detection unit, streamlined the operation process and reduced the exposure risk of directly handling cryptococcal samples. It did not require any additional instruments and demonstrated a rapid, reliable, as well as high-efficiency approach. It truly realized the “sample-to-answer” application and could be easily used for clinical cryptococcal prediagnosis.

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Synergistic use of electroosmotic flow and magnetic forces for nucleic acid extraction

The Analyst ◽

10.1039/c9an02191d ◽

2020 ◽

Vol 145 (6) ◽

pp. 2412-2419 ◽

Cited By ~ 1

Author(s):

Rachel N. Deraney ◽

Lindsay Schneider ◽

Anubhav Tripathi

Keyword(s):

Nucleic Acid ◽

Electric Field ◽

Microfluidic Chip ◽

Applied Electric Field ◽

Electroosmotic Flow ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Magnetic Forces ◽

Extraction And Purification

NA extraction and purification utilitzing a microfluidic chip with applied electric field to induce electroosmotic flow opposite the magnetic NA-bound bead mix.

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A point of care platform based on microfluidic chip for nucleic acid extraction in less than 1 minute

Biomicrofluidics ◽

10.1063/1.5088552 ◽

2019 ◽

Vol 13 (3) ◽

pp. 034102 ◽

Cited By ~ 4

Author(s):

Jianzhong Zhang ◽

Xiaosong Su ◽

Jiasu Xu ◽

Jin Wang ◽

Juntian Zeng ◽

...

Keyword(s):

Nucleic Acid ◽

Microfluidic Chip ◽

Point Of Care ◽

Acid Extraction ◽

Nucleic Acid Extraction

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Solid phase nucleic acid extraction technique in a microfluidic chip using a novel non-chaotropic agent: dimethyl adipimidate

Lab on a Chip ◽

10.1039/c3lc51035b ◽

2014 ◽

Vol 14 (2) ◽

pp. 359-368 ◽

Cited By ~ 23

Author(s):

Yong Shin ◽

Agampodi Promoda Perera ◽

Chee Chung Wong ◽

Mi Kyoung Park

Keyword(s):

Nucleic Acid ◽

Microfluidic Chip ◽

Solid Phase ◽

Extraction Technique ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Chaotropic Agent

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Rapid detection of foot-and-mouth disease virus using a field-portable nucleic acid extraction and real-time PCR amplification platform

The Veterinary Journal ◽

10.1016/j.tvjl.2011.10.017 ◽

2012 ◽

Vol 193 (1) ◽

pp. 67-72 ◽

Cited By ~ 24

Author(s):

Mikidache Madi ◽

Andrea Hamilton ◽

David Squirrell ◽

Valerie Mioulet ◽

Phillip Evans ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Disease Virus ◽

Real Time Pcr ◽

Rapid Detection ◽

Pcr Amplification ◽

Foot And Mouth Disease ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Mouth Disease Virus

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Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

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Forensic evaluation of two nucleic acid extraction systems and validation of a RT-qPCR protocol for identification of SARS-CoV-2 in post-mortem nasopharyngeal swabs

Forensic Science International ◽

10.1016/j.forsciint.2021.110775 ◽

2021 ◽

pp. 110775

Author(s):

Pedro A. Barrio ◽

Amparo Fernández-Rodríguez ◽

Pablo Martín ◽

Coro Fernández ◽

Lourdes Fernández ◽

...

Keyword(s):

Nucleic Acid ◽

Forensic Evaluation ◽

Acid Extraction ◽

Post Mortem ◽

Nucleic Acid Extraction

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Evaluation of an Automated High-Throughput Liquid-Based RNA Extraction Platform on Pooled Nasopharyngeal or Saliva Specimens for SARS-CoV-2 RT-PCR

Viruses ◽

10.3390/v13040615 ◽

2021 ◽

Vol 13 (4) ◽

pp. 615

Author(s):

Allen Wing-Ho Chu ◽

Cyril Chik-Yan Yip ◽

Wan-Mui Chan ◽

Anthony Chin-Ki Ng ◽

Dream Lok-Sze Chan ◽

...

Keyword(s):

Nucleic Acid ◽

High Throughput ◽

Rna Extraction ◽

Nasopharyngeal Swab ◽

Acid Extraction ◽

Rt Pcr ◽

Nucleic Acid Extraction ◽

Suitable Alternative ◽

Pcr Inhibitors ◽

Pcr Targeting

SARS-CoV-2 RT-PCR with pooled specimens has been implemented during the COVID-19 pandemic as a cost- and manpower-saving strategy for large-scale testing. However, there is a paucity of data on the efficiency of different nucleic acid extraction platforms on pooled specimens. This study compared a novel automated high-throughput liquid-based RNA extraction (LRE) platform (PHASIFYTM) with a widely used magnetic bead-based total nucleic acid extraction (MBTE) platform (NucliSENS® easyMAG®). A total of 60 pools of nasopharyngeal swab and 60 pools of posterior oropharyngeal saliva specimens, each consisting of 1 SARS-CoV-2 positive and 9 SARS-CoV-2 negative specimens, were included for the comparison. Real-time RT-PCR targeting the SARS-CoV-2 RdRp/Hel gene was performed, and GAPDH RT-PCR was used to detect RT-PCR inhibitors. No significant differences were observed in the Ct values and overall RT-PCR positive rates between LRE and MBTE platforms (92.5% (111/120] vs 90% (108/120]), but there was a slightly higher positive rate for LRE (88.3% (53/60]) than MBTE (81.7% (49/60]) among pooled saliva. The automated LRE method is comparable to a standard MBTE method for the detection of SAR-CoV-2 in pooled specimens, providing a suitable alternative automated extraction platform. Furthermore, LRE may be better suited for pooled saliva specimens due to more efficient removal of RT-PCR inhibitors.

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