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Device Technologies and Biomedical Robotics

CENTRIFUGE-FREE DRYING OF SILICA MEMBRANE FOR HIV RNA EXTRACTION FROM WHOLE BLOOD FOR SMARTPHONE-BASED DETECTION

(K-404) CENTRIFUGE-FREE DRYING OF SILICA MEMBRANE FOR HIV RNA EXTRACTION FROM WHOLE BLOOD FOR SMARTPHONE-BASED DETECTION

Saturday, October 14, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row K - Poster # 404

Presenting Author(s)

Mia T. Nair

Undergraduate Research Assistant
Purdue University
West Lafayette, Indiana, United States

Co-Author(s)

WN

Winston Ngo (he/him/his)

Undergraduate Research Assistant
Purdue University
West Lafayette, Indiana, United States

Last Author(s)

Emeka Nwanochie

Research Assistant (PhD Candidate)
Purdue University
WEST LAFAYETTE, Indiana, United States

Primary Investigator(s)

JL

Jacqueline C. Linnes, PhD (she/her/hers)

Marta E. Gross Associate Professor
Purdue University
West Lafayette, Indiana, United States

Introduction:: As of the end of 2022, around 39.0 million people worldwide were living with HIV 1. By 2030, UNAIDS hopes that 95% of people receiving antiretroviral therapy (ART) achieve viral suppression; however, in 2021, only 32% had reached viral suppression 2. Monitoring viral load is crucial for tracking adherence to the treatment, diagnosing HIV, and achieving global epidemic control 2 3. Patients who achieved viral suppression through ART have lower transmission rates and fewer health care visits 2. There is a need for user-friendly viral load testing that is portable for community health workers to reach patients on ART, and easy to implement in resource-limited areas. Demonstrated here is an innovative method that utilizes a portable hand-held bulb pump to achieve rapid sample preparation from whole blood using a commercial RNA extraction kit. The extraction method is essential because while RT-LAMP is effective, it lacks the sensitivity to pick up very low copies of HIV RNA. The extracted sample is of sufficient quality for RT-PCR in tube and RT-LAMP on-chip.

Materials and Methods:: HIV RNA was extracted from HIV-spiked blood at a concentration of 1000 virion copies per microliter. We utilized spin columns and lysis, wash, and elution buffers from a commercial kit (#52904, QIAamp Viral RNA Kit). The process involved lysis of the whole blood, followed by washing and elution steps. In detail, the lysed sample was carefully transferred using a pipette into a spin column connected to a tube, through which negative pressure was applied using a syringe, facilitating the passage of the sample through the column (Figure 1A). Subsequently, the bound HIV RNA was subjected to washing, including an additional third wash with 100% acetone. To expedite the drying process, we 3D-printed a device that was connected to a portable hand-held pump (Figure 1B). This device supplied a constant air flow directly onto the membrane for one minute, promoting rapid drying. The drying process is a critical step in the extraction process as the acetone aids in washing away the residual chaotropic salts and ethanol from the wash buffers which facilitates in eluting of the RNA. Finally, the HIV RNA was eluted from the membrane and then amplified through RT-PCR and RT-LAMP reactions (Figure 1C).

Results, Conclusions, and Discussions::

RESULTS AND DISCUSSION

The HIV RNA was effectively extracted from whole blood samples yielding results that were comparable to those obtained following the centrifuge-processed samples. Cycle threshold (CT) values obtained from the centrifuge-processed samples averaged at 33.48 with a standard deviation of 0.36 whereas samples processed without a centrifuge but with the drying step that included the bulb pump averaged around 33.91 with a standard deviation of 0.68.

CONCLUSION

We have demonstrated an equipment-free rapid HIV RNA extraction method utilizing a portable bulb pump membrane drying step. Future work includes determining the limit of detection and a comparative analysis of other drying methods.

Acknowledgements (Optional): :

References (Optional): :

REFERENCES

World Health Organization. (n.d). The Global Health Observatory - HIV. World Health Organization. https://www.who.int/data/gho/data/themes/hiv-aids

Lecher, S. L., et al. (2021, May 28). HIV viral load monitoring among patients receiving antiretroviral therapy - eight Sub-Saharan Africa countries, 2013-2018. MMWR. Morbidity and mortality weekly report. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158895/

Oxford Academic. (n.d). HIV Viral Load Monitoring in Resource-Limited Regions: Optional or Necessary?. Clinical Infectious Diseases. https://academic.oup.com/cid/article/44/1/128/431117