HIV Detection from Human Serum with Paper-Based Isotachophoretic RNA Extraction and Reverse Transcription Recombinase Polymerase Amplification

<p>The number of people living with HIV continues to increase with the current total near 38 million, of which about 26 million are receiving antiretroviral therapy. These treatment regimens are highly effective when properly managed, requiring routine viral load monitoring to assess successful viral suppression. Efforts to expand access by decentralizing HIV nucleic acid testing in low- and middle-income countries has been hampered by the cost and complexity of current tests. Sample preparation of blood samples has traditionally relied on cumbersome RNA extraction methods, and it continues to be a key bottleneck for developing low-cost POC nucleic acid tests. We present a microfluidic paper-based analytical device (µPAD) for extracting RNA and detecting HIV in serum, leveraging low-cost materials, simple buffers, and an electric field. We detect HIV virions and MS2 bacteriophage internal control in human serum using a novel lysis and RNase inactivation method, paper-based isotachophoresis (ITP) for RNA extraction, and duplexed reverse transcription recombinase polymerase amplification (RT-RPA) for nucleic acid amplification. We design a specialized ITP system to extract and concentrate RNA, while excluding harsh reagents used for lysis and RNase inactivation. We found the ITP µPAD can extract and purify 5,000 HIV RNA copies per mL of serum. We then demonstrate detection of HIV virions and MS2 bacteriophage in human serum within 45-minutes.</p>

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HIV detection from human serum with paper-based isotachophoretic RNA extraction and reverse transcription recombinase polymerase amplification

The Analyst ◽

10.1039/d0an02483j ◽

2021 ◽

Author(s):

Andrew T. Bender ◽

Benjamin P. Sullivan ◽

Jane Zhang ◽

David C Juergens ◽

Lorraine Lillis ◽

...

Keyword(s):

Antiretroviral Therapy ◽

Human Serum ◽

Reverse Transcription ◽

Rna Extraction ◽

Recombinase Polymerase Amplification ◽

People Living With Hiv ◽

Treatment Regimens ◽

Living With Hiv

The number of people living with HIV continues to increase with the current total near 38 million, of which about 26 million are receiving antiretroviral therapy (ART). These treatment regimens...

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Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler

10.1101/2020.04.17.042366 ◽

2020 ◽

Cited By ~ 9

Author(s):

Soon Keong Wee ◽

Suppiah Paramalingam Sivalingam ◽

Eric Peng Huat Yap

Keyword(s):

Nucleic Acid ◽

Reverse Transcription ◽

Rna Extraction ◽

Cost Effective ◽

Turnaround Time ◽

Nucleic Acid Amplification ◽

Detection Methods ◽

N Gene ◽

Direct Pcr ◽

Viral Lysis

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as 6 RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification and detection are achieved in a single-tube homogeneous reaction within 36 minutes. This minimized hands-on time, reduces turnaround-time for sample-to-result and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening and research in countries and regions where laboratory capabilities are limiting.

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Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler

Genes ◽

10.3390/genes11060664 ◽

2020 ◽

Vol 11 (6) ◽

pp. 664 ◽

Cited By ~ 12

Author(s):

Soon Keong Wee ◽

Suppiah Paramalingam Sivalingam ◽

Eric Peng Huat Yap

Keyword(s):

Nucleic Acid ◽

Reverse Transcription ◽

Rna Extraction ◽

Cost Effective ◽

Turnaround Time ◽

Nucleic Acid Amplification ◽

Detection Methods ◽

N Gene ◽

Direct Pcr ◽

Viral Lysis

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.

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Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring

Molecules ◽

10.3390/molecules26216617 ◽

2021 ◽

Vol 26 (21) ◽

pp. 6617

Author(s):

Eva Rajh ◽

Tina Šket ◽

Arne Praznik ◽

Petra Sušjan ◽

Alenka Šmid ◽

...

Keyword(s):

Oral Cavity ◽

Reverse Transcription ◽

Screening Program ◽

Quantitative Polymerase Chain Reaction ◽

Rna Extraction ◽

Diagnostic Sensitivity ◽

Nucleic Acid Amplification ◽

Nasopharyngeal Swab ◽

Viral Spread ◽

One Step

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

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Rapid Detection of SARS-CoV-2 Using Reverse transcription RT-LAMP method

10.1101/2020.03.02.20030130 ◽

2020 ◽

Cited By ~ 29

Author(s):

Weihua Yang ◽

Xiaofei Dang ◽

Qingxi Wang ◽

Mingjie Xu ◽

Qianqian Zhao ◽

...

Keyword(s):

Nucleic Acid ◽

Reverse Transcription ◽

Virus Disease ◽

Lamp Assay ◽

Nucleic Acid Amplification ◽

N Gene ◽

Nucleic Acid Detection ◽

Lamp Method ◽

Rt Pcr ◽

Rapid Amplification

AbstractCorona Virus Disease 2019 (COVID-19) is a recently emerged life-threatening disease caused by SARS-CoV-2. Real-time fluorescent PCR (RT-PCR) is the clinical standard for SARS-CoV-2 nucleic acid detection. To detect SARS-CoV-2 early and control the disease spreading on time, a faster and more convenient method for SARS-CoV-2 nucleic acid detecting, RT-LAMP method (reverse transcription loop-mediated isothermal amplification) was developed. RNA reverse transcription and nucleic acid amplification were performed in one step at 63 °C isothermal conditions, and the results can be obtained within 30 minutes. ORF1ab gene, E gene and N gene were detected at the same time. ORF1ab gene was very specific and N gene was very sensitivity, so they can guarantee both sensitivity and specificity for SARS-CoV-2. The sensitivity of RT-LAMP assay is similar to RT-PCR, and specificity was 99% as detecting 208 clinical specimens. The RT-LAMP assay reported here has the advantages of rapid amplification, simple operation, and easy detection, which is useful for the rapid and reliable clinical diagnosis of SARS-CoV-2.

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Amplification-Free DNA Detection Using a Microtip-Sensor Decorated With LNA Probes for Rapid TB Screening

Volume 2: Biomedical and Biotechnology Engineering; Nanoengineering for Medicine and Biology ◽

10.1115/imece2011-64378 ◽

2011 ◽

Author(s):

Shinnosuke Inoue ◽

Woon-Hong Yeo ◽

Jong-Hoon Kim ◽

Jae-Hyun Chung ◽

Kyong-Hoon Lee ◽

...

Keyword(s):

Nucleic Acid ◽

Staphylococcus Epidermidis ◽

Genomic Dna ◽

Bacterial Culture ◽

Low Cost ◽

Surrogate Marker ◽

High Sensitivity ◽

Diagnostic Methods ◽

Nucleic Acid Amplification ◽

Highly Sensitive

Tuberculosis (TB) is an epidemic affecting one-third of the world’s population, mostly in developing and low-resource settings. People having active pulmonary TB are considered highly infectious; therefore, it is critical to identify and treat these patients rapidly before spreading to others. However, the most reliable TB diagnostic methods of bacterial culture or nucleic acid amplification are time-consuming and expensive. The challenge of TB diagnosis lies in highly sensitive and specific screening with low cost. Here, we present an LNA-modified microtip-sensor, which is capable of selectively detecting low-abundance DNA from bacteria. When genomic DNA of Bacillus Calmette-Gue´rin (BCG, a surrogate marker of Mycobacterium bovis), and genomic DNA of Staphylococcus epidermidis (S. epi) are used, the microtip-sensor yields the detection limit of 1,000 copies/mL within 20 minutes. The high sensitivity and specificity approaching nucleic acid amplification methods can potentially overcome the current challenges for rapid TB screening.

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A Simple, Low-Cost Platform for Real-Time Isothermal Nucleic Acid Amplification

Sensors ◽

10.3390/s150923418 ◽

2015 ◽

Vol 15 (9) ◽

pp. 23418-23430 ◽

Cited By ~ 14

Author(s):

Pascal Craw ◽

Ruth Mackay ◽

Angel Naveenathayalan ◽

Chris Hudson ◽

Manoharanehru Branavan ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Low Cost ◽

Nucleic Acid Amplification ◽

Isothermal Nucleic Acid Amplification

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Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acid Amplification for Detecting Acute HIV Infection

Clinical Chemistry ◽

10.1373/clinchem.2015.243287 ◽

2015 ◽

Vol 61 (11) ◽

pp. 1372-1380 ◽

Cited By ~ 18

Author(s):

Carlos Cabrera ◽

Lei Chang ◽

Mars Stone ◽

Michael Busch ◽

David H Wilson

Keyword(s):

Nucleic Acid ◽

Early Detection ◽

Hiv Infection ◽

Single Molecule ◽

Low Cost ◽

Nucleic Acid Amplification ◽

Analytical Sensitivity ◽

Acute Hiv Infection ◽

Acute Hiv ◽

Viral Isolates

Abstract BACKGROUND Nucleic acid testing (NAT) has become the standard for high sensitivity in detecting low levels of virus. However, adoption of NAT can be cost prohibitive in low-resource settings where access to extreme sensitivity could be clinically advantageous for early detection of infection. We report development and preliminary validation of a simple, low-cost, fully automated digital p24 antigen immunoassay with the sensitivity of quantitative NAT viral load (NAT-VL) methods for detection of acute HIV infection. METHODS We developed an investigational 69-min immunoassay for p24 capsid protein for use on a novel digital analyzer on the basis of single-molecule-array technology. We evaluated the assay for sensitivity by dilution of standardized preparations of p24, cultured HIV, and preseroconversion samples. We characterized analytical performance and concordance with 2 NAT-VL methods and 2 contemporary p24 Ag/Ab combination immunoassays with dilutions of viral isolates and samples from the earliest stages of HIV infection. RESULTS Analytical sensitivity was 0.0025 ng/L p24, equivalent to 60 HIV RNA copies/mL. The limit of quantification was 0.0076 ng/L, and imprecision across 10 runs was <10% for samples as low as 0.09 ng/L. Clinical specificity was 95.1%. Sensitivity concordance vs NAT-VL on dilutions of preseroconversion samples and Group M viral isolates was 100%. CONCLUSIONS The digital immunoassay exhibited >4000-fold greater sensitivity than contemporary immunoassays for p24 and sensitivity equivalent to that of NAT methods for early detection of HIV. The data indicate that NAT-level sensitivity for acute HIV infection is possible with a simple, low-cost digital immunoassay.

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