Compact Rapid Detection Kit to Test for Monkeypox Virus Shows Promising Results

In a recent report in the journal Travel Medicine and Infectious Disease, researchers introduced a compact rapid detection kit that tests for monkeypox virus using a combination of Recombinase Polymerase Amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology.

Background

The monkeypox outbreak has spread widely outside the endemic west and central African countries during 2022, and the World Health Organization (WHO) has now declared it a Public Health Emergency of International Concern. The etiological agent of monkeypox is the monkeypox virus belonging to the Orthopoxvirus genus.

The currently used test for detecting monkeypox is the polymerase chain reaction test (PCR), which amplifies Orthopoxvirus or specific monkeypox virus deoxyribonucleic acid (DNA). However, PCR is a tedious process requiring a thermocycler, trained personnel, and a laboratory.

The early detection and containment of infected individuals are imperative for controlling a disease outbreak. Immunoassays such as lateral flow strips are simpler and faster than PCRs but have higher cross-reactivity for orthopoxviruses, making the precise detection of monkeypox difficult. Therefore, a technology that rapidly detects monkeypox and provides accurate and reliable results is essential.

Detection mechanism

In the present study, the researchers developed a rapid detection kit or pocket lab to test for the monkeypox virus. The detection kit uses the principles of RPA and CRISPR technology to increase the specificity and sensitivity of the analysis. The RPA amplifies target genes of the monkeypox virus, which are scanned by the CRISPR guide ribonucleic acid (RNA) and the CRISPR-associated protein 12a (Cas12a) enzyme.

The CRISPR/Cas12sa-mediated cleavage of the sequence occurs only when the target monkeypox amplicons are detected, eliminating the possibility of non-specific signals. The cleavage results in fluorescence, indicating monkeypox-positive samples.

Pocket lab

The kit weighed 500 g, and the case was water-resistance and compact enough to fit inside a pocket. The components comprised two three-dimensional printed heating blocks — block A, which heats the samples to 80 °C for viral shell lysis, and block B, which heats the samples to 40 °C for DNA amplification.

The kit contained tubes with trehalose-protected lyophilized enzymes and sampling tubes with sodium chloride and magnesium acetate solution. A rehydration buffer containing primers and the fluorescent reporter, and an ultraviolet flashlight (UV) to detect the fluorescence were also included in the kit.

The testing process involves adding the sample to the sampling tube and using a heating block A to lyse the viral shell. The heated sample is added to the tube containing lyophilized enzymes, to which the rehydration buffer is added. Heating block B is then employed to amplify the target region. Finally, the UV flashlight is used to detect the fluorescence.

During the trial test, the researchers ran using pseudotyped monkeypox virus samples, the total time taken was 25 minutes, and samples with viral particles as low as ten were detected with accuracy.

Conclusions

To summarize, the researchers introduced a novel rapid detection kit that was compact and could be used to detect the monkeypox virus without the use of complicated instruments, trained technicians, or a laboratory set-up.

The detection kit uses the principles of RPA and CRISPR to amplify target regions of the monkeypox DNA and cleave it using the CRISPR/Cas12a complex, with a fluorescent reporter signaling the detection of monkeypox viral DNA.

The trials with the pseudotyped virus indicated successful and rapid detection of the monkeypox virus, suggesting the kit’s potential use for rapid testing of travelers and in areas that lack appropriate medical infrastructure and trained staff.

Journal reference:

Wei, J., Meng, X., Li, J., & Pang, B. (2022). Pocket lab for the rapid detection of monkeypox virus. Travel Medicine and Infectious Disease. 

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A Rapid Test that Detects Group B Streptococcus is in Development

Scientists at the University of Bath are looking for volunteers to help them develop a new test that could prevent dangerous bacterial infections in newborn babies which currently kills one baby every week in the UK.

Between 20-40 per cent of women carry a bacterium called Group B Streptococcus (GBS) in their vaginas.

This normally does not cause harm, but during pregnancy, it can be passed onto the baby, which sometimes causes life-threatening infections and can result in life-long disabilities or death.

GBS is currently the leading cause of neonatal infection – in the UK, it kills around one newborn baby a week and leaves another permanently disabled.

Professor Toby Jenkins, from the University of Bath’s Department of Chemistry, is leading a team to develop a new rapid detection system that can test mothers for GBS during labour, identifying those at risk of infection so they can be treated with antibiotics and therefore reduce the risk of the baby becoming infected.

He said: “The current test is done at 36-38 weeks of pregnancy, but this may not reflect the situation during labour, resulting in either risk to the baby, or unnecessary antibiotic use by the mother.

“Our test aims to be much faster – with results in under 45 minutes – and can be done during established labour so the result is more accurate.

“If the mother is found to have GBS at this stage, antibiotics can be immediately offered, which is an effective way of preventing transmission.”

The test consists of a vaginal swab taken from the mother which is placed in a liquid that can detect GBS.

Professor Jenkins added: “We are now looking for participants to donate two vaginal swabs so we can determine the efficacy, sensitivity and specificity of the test to GBS. Participants are completely anonymous, but would be hugely helpful to our research project.”

The team is looking for individuals born biologically female, who are over 18 years old, currently not pregnant and able to take their own samples to send anonymously to the lab.

Participation is entirely confidential: the team will not know the identity of donors.

The study has full ethics approval from REACH, is supported by the charity Group B Strep Support, and funded by the European Union Horizon 2020 project STIMULUS.

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Nanotech Bubbles Burst When They Detect Viruses in the Air

Scientists have shown that they can detect SARS-CoV-2, the virus that causes COVID-19, in the air by using a nanotechnology-packed bubble that spills its chemical contents like a broken piñata when encountering the virus.

Such a detector could be positioned on a wall or ceiling, or in an air duct, where there’s constant air movement, to alert occupants immediately when even a trace level of the virus is present.

The heart of the nanotechnology is a micelle, a molecular structure composed of oils, fats and sometimes water with inner space that can be filled with air or another substance. Micelles are often used to deliver anticancer drugs in the body and are a staple in soaps and detergents. Almost everyone has encountered a micelle in the form of soap bubbles.

A team of scientists at the Department of Energy’s Pacific Northwest National Laboratory created a new kind of micelle, one that is stamped on the surface with copies of an imprinted particle for SARS-CoV-2.

The team filled micelles with a salt capable of creating an electronic signal but that is quiescent when packed inside a micelle. When a viral particle interacts with one of the imprinted receptors on the surface, the micelle pops open, spilling the salt and sending out an electronic signal instantly.

The system acts like a signal magnifier, translating the presence of one viral particle into 10 billion molecules that together create a detectable signal. The developers say that the detector has advantages over today’s technologies; it produces a signal faster, requires a much lower level of viral particles, or produces fewer errors.

“There is a need for this kind of low-cost detection system,” said PNNL scientist Lance Hubbard, a nanotechnology specialist and an author of the paper. “Perhaps it could be implemented in schools, or in hospitals or emergency rooms before patients have been fully assessed—anywhere you need to know immediately that the virus is present.”

PNNL’s micelle technology is the product of an arduous chain of 279 separate chemical steps developed by first author Samuel Morrison together with Hubbard and other PNNL scientists.

Detecting One Viral Particle Out of Billions

The team estimates that the technology can pluck one viral particle out of billions of other particles. The detector is so sensitive that the team had a challenging time identifying the lower limit. The team used both inactivated SARS-CoV-2 viral particles and the virus’s spike protein in its tests.

While the technology detects the virus within a millisecond, the device takes an additional minute to run quality-control software to confirm the signal and prevent false alarms.

Micelles can be delicate, like a soap bubble from a child’s wand. But, under certain circumstances, scientists can make hardier micelles that spill their contents at just the right time and place—for instance, these micelles that burst open when a viral particle is detected.

The PNNL micelle is bilayer, with one polymer-coated micelle inside the other, and the entire structure immersed in water. Each micelle is about 5 microns wide. On the outer surface are several imprinted particles, made of silica, about 500 nanometers wide. Each imprint is an opportunity for a COVID-causing viral particle to bind, causing the bilayer micelle to pop open.

“Combining micelles with a technology to imprint or stamp them is not something many people have done before,” said Hubbard. “Imprinting a molecule with our molecule of interest inserts a vulnerability into the micelle—which is what we want in this case.”

Morrison, a former Marine, began this line of work hoping to develop a new way to help soldiers quickly detect explosives in combat. He connected with Hubbard, an expert in nanosynthesis. They switched the focus of the project to SARS-CoV-2 when the pandemic hit. Other possible uses of the technology include detection of fentanyl and environmental toxins.

Battelle, which manages and operates PNNL for DOE, has filed for a patent on the technology. The scientists say the technology needs to be developed further, perhaps with a licensing partner, before it can be deployed broadly.

Reference

Detection of SARS-COV-2 by functionally imprinted micelles. MRS Communications, 25 October 2022

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Face Mask Detects Viral Exposure

Scientists have created a face mask that can detect common respiratory viruses, including influenza and the coronavirus, in the air in droplets or aerosols. The highly sensitive mask, presented September 19 in the journal Matter, can alert the wearers via their mobile devices within 10 minutes if targeted pathogens are present in the surrounding air.

"Previous research has shown face mask wearing can reduce the risk of spreading and contracting the disease. So, we wanted to create a mask that can detect the presence of virus in the air and alert the wearer," says Yin Fang, the study's corresponding author and a material scientist at Shanghai Tongji University.

Respiratory pathogens that cause COVID-19 and H1N1 influenza spread through small droplets and aerosols released by infected people when they talk, cough, and sneeze. These virus-containing molecules, especially tiny aerosols, can remain suspended in the air for a long time.

Fang and his colleagues tested the mask in an enclosed chamber by spraying the viral surface protein containing trace-level liquid and aerosols on the mask. The sensor responded to as little as 0.3 microliters of liquid containing viral proteins, about 70 to 560 times less than the volume of liquid produced in one sneeze and much less than the volume produced by coughing or talking, Fang says.

The team designed a small sensor with aptamers, which are a type of synthetic molecule that can identify unique proteins of pathogens like antibodies. In their proof-of-concept design, the team modified the multi-channel sensor with three types of aptamers, which can simultaneously recognize surface proteins on SARS-CoV-2, H5N1, and H1N1.

Once the aptamers bind to the target proteins in the air, the ion-gated transistor connected will amplify the signal and alert the wearers via their phones. An ion-gated transistor is a novel type of device that is highly sensitive, and thus the mask can detect even trace levels of pathogens in the air within 10 minutes.

"Our mask would work really well in spaces with poor ventilation, such as elevators or enclosed rooms, where the risk of getting infected is high," Fang says. In the future, if a new respiratory virus emerges, they can easily update the sensor's design for detecting the novel pathogens, he adds.

Next, the team hopes to shorten the detection time and further increase the sensitivity of the sensor by optimizing the design of the polymers and transistors. They are also working on wearable devices for a variety of health conditions including cancers and cardiovascular diseases.

"Currently, doctors have been relying heavily on their experiences in diagnosing and treating diseases. But with richer data collected by wearable devices, disease diagnosis and treatment can become more precise," Fang says.

The work is supported by National Key Research and Development Program, National Natural Science Foundation of China, Science and Technology Commission of Shanghai Municipality, Shanghai Municipal Science and Technology Major Project and the Fundamental Research Funds for the Central Universities.

Journal Reference

Bingfang Wang, Deqi Yang, Zhiqiang Chang, Ru Zhang, Jing Dai, Yin Fang. Wearable bioelectronic masks for wireless detection of respiratory infectious diseases by gaseous media. Matter, 2022.

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Blood-Based Diagnostic Cuts Time to Infection Detection

U.S researchers have developed a rapid processing system that can dramatically improve the time taken to detect bacterial and fungal infections in the bloodstream, potentially allowing faster administration of antibiotics.

The culture-free, “biphasic” approach enables pathogen DNA to be amplified directly from just 1 ml of whole blood, reducing the time needed for a result to less than 2.5 hours compared with more than 20 hours using conventional methods.

The system offers single-molecule sensitivity in detecting pathogens including methicillin-resistant and methicillin-sensitive Staphylococcus aureus, Escherichia coli, and Candida albicans.

Validation studies using 63 whole-blood samples showed total agreement in sensitivity and specificity with clinical laboratory results that used blood culture and polymerase chain reaction (PCR).

The new system, described in the Proceedings of the National Academy of Sciences, involves rapidly drying blood and creating a porous microfluidic and nanofluidic network within this.

DNA amplification enzymes and primers are then able to diffuse into the dried blood matrix to access any pathogen DNA within, initiating its amplification without the need for conventional nucleic acid purification.

Researcher Rashid Bashir, professor of bioengineering at the University of Illinois at Urbana-Champaign, U.S.A., told Inside Precision Medicine that traditional blood culture could take many days to provide enough bacteria for subsequent PCR detection.

“Alternatively, our approach using a new blood drying technique can be used to detect pathogens in less than a few hours. This can potentially be very important for rapid and early detection of onset of sepsis caused by bacteremia.”

Antibiotic therapy within three hours of initial, symptom-based recognition can significantly reduce the risk of death from bloodstream infections and bacteremia, the researchers note.

However, the current clinical gold standard for diagnosing sepsis and bloodstream infections remains blood culture followed by nucleic acid amplification and detection using PCR.

“The blood culture step is too slow and cumbersome to allow for initial management of patients and thus contributes to high mortality,” the authors explain.

“Moreover, in the absence of timely results from robust diagnostic tests, the patients are administered highly potent broad-spectrum antibiotics without any patient stratification, increasing antimicrobial resistance and emergence of drug-resistant and atypical pathogens.”

The new platform uses whole blood, which can be dried in as little as 10 minutes using high temperatures of 95 °C. The dried blood then acts as a substrate that does not take part in the reaction, and inhibitory elements such as platelets, cells and proteins are neutralized to form part of this substrate.

Thermal lysis improves the porosity of microfluidic and nanofluidic networks within the dried blood matrix, which allows enzymes to access pathogen DNA and initiate amplification with single-molecule sensitivity, thereby bypassing the need for conventional DNA purification.

The dried blood solid phase does not re-mix with the supernatant and keeps the high heme locked in red blood cells in the background while amplicons diffuse out and bind to fluorescent dye in the clear supernatant phase, leading the researchers to term this “biphasic amplification”.

Reporting their findings, they propose: “The reduction in instrumentation complexity and costs compared to blood culture and alternate molecular diagnostic platforms can have broad applications in healthcare systems in developed world and resource-limited settings.”

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