Study: CRISPR/Cas12a-mediated ultrasensitive and on-site monkeypox viral testing. Image Credit: Dotted Yeti / Shutterstock

The monkeypox virus (MPXV), which was first isolated in 1958, is a double-stranded DNA (dsDNA) virus. It was recognized as a zoonotic disease when the first human case was detected in the Democratic Republic of the Congo in 1970. There is currently a global concern over MPXV due to its wider transmission from Central and West Africa.

Rapid, ultrasensitive, and specific detection is essential to curb this virus's spread. In a new study posted to the medRxiv* preprint server, researchers in China developed an MPXV assay combining clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR/Cas) and recombinase-aided amplification (RAA) for the first time. The assay exhibited high selectivity and could distinguish between MPXV and other orthopoxviruses.

Background

Currently, MPXV testing uses methods such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and loop-mediated isothermal amplification (LAMP). PCR is the gold standard and is accurate and sensitive but challenging to deploy in low-resource areas. ELISA could provide false positive results for recent or remote vaccination, and LAMP’s disadvantages are related to a complicated primer design, poor quantitative performance, etc. Rapid, ultrasensitive, and low-cost methods facilitating on-site and facile MPXV detection have remained absent.

About the Study

CRISPR/Cas was first discovered in the adaptive immune system of prokaryotes. CRISPR/Cas12a system integrates signal transduction, and biorecognition has been used to detect several viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It has many advantages related to mild conditions, high sensitivity, operational ease, and powerful signal amplification.

To date, critical issues related to using CRISPR technology in MPXV detection, such as probe screening, analytical performance, pre-amplification, and point-of-care testing (POCT), have not been reported. Therefore, the current study proposed a rapid and ultrasensitive assay combining recombinase-aided amplification (RAA) and CRISPR/Cas12a, i.e., the RAA-Cas12a-MPXV assay, for the first time. The principle comprised three steps: RAA amplification, CRISPR/Cas12a cleavage, and signal output.

Key Results

In the first step, RAA selecting the DNA template produced a large number of amplicons. This augmented the sensitivity of the assay significantly. In the second step, the trans-cleavage activity of Cas12a was activated, which resulted in numerous ssDNA reporters being cleaved. The last step produced two different signal output modes: fluorescence assay for FQ reporters and lateral flow strip, which improved the usability and suitability of the RAA-Cas12a-MPXV assay.

Fluorescence results with or without DNA templates and the RAA product were compared to evaluate the feasibility of the assay. The no DNA group was the control, which showed no significant fluorescence change. This indicated that, in the absence of DNA templates, no RAA amplicon was generated for it to be recognized by subsequent CRISPR/Cas12a.

To ensure greater selectivity and sensitivity, three pairs of primers that bound to various sites of the MPXV-specific F3L gene were designed. To screen the optimal primers for future experiments, the RAA-Cas12a-MPXV fluorescence assay and the agarose gel electrophoresis (AGE) were performed. The third pair (F2+R2) achieved the brightest specific amplification band.

Many important experimental conditions related to the CRISPR/Cas12a system and the RAA-Cas12a-MPXV assay were optimized, with RAA's temperature and reaction time being the first. Next, the CRISPR/Cas12a system with crRNA2 was optimized.

The RAA-Cas12a-MPXV fluorescence assay was deemed to be easy to operate and amenable to be used for rapid testing. Compared to the PCR-Cas12a-MPXV fluorescence assay, the RAA-Cas12a-MPXV fluorescence assay revealed outstanding sensitivity with a 1000-fold lower limit of detection (LOD).

The selectivity of the RAA-Cas12a-MPXV fluorescence assay was determined by comparing the degree of fluorescence produced by MPXV with other viruses, such as variola virus (VARV), cowpox virus (CPXV), severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), Toxoplasma Gondii virus (TOXV), and African swine fever virus (ASFV). Both naked eye observation and fluorescence values indicated that only MPXV induced enhanced fluorescence, while other viruses did not.

The FAM-Biotin reporter (FB-reporter) reporter was designed as a substitute for the FQ reporter of the fluorescence assay, and subsequently, the RAA-Cas12a-MPXV lateral flow strip assay was established for POCT. The addition of a reaction solution containing intact FB reporters cracked FAMs and biotin in the sample pads caused rapid binding of the anti-FAM antibody/AuNP complexes with isolated FAMs and FB reporters as the sample migrated forward. Finally, streptavidin, immobilized at the control band, captured cracked biotins and the FB reporter/anti-FAM antibody/AuNP complexes.

Initially, the control band appeared red due to the aggregation of AuNPs. Subsequently, the rest of the isolated FAM/anti-FAM antibody/AuNP complexes traveled towards the test band and reacted with FAM antibodies appearing red. Finally, amplicons activated Cas12a to cleave all FB reporters. The change of color only remained at the test band.

In a nutshell, when the color change was observed only in the test band or both the control and the test band, it was considered a positive result. In contrast, if the color change was only found at the control band, it indicated a negative result, i.e., the absence of DNA templates led to no amplicons for Cas12a activation. 

Conclusions

A vital advantage of the RAA-Cas12a-MPXV assay is that it can be conducted at a mild temperature compared to conventional processes. Importantly, this assay was a powerful MPXV diagnosis method with superior selectivity, sensitivity, and portability.  By Dr. Priyom Bose, Ph.D.Oct 18 2022 Reviewed by Danielle Ellis, B.Sc. Source Libyan Cloud Agency

By URN

After a 3-day effort, police working with the National Children Authority and the ministry of Education officials have identified the school whose infant learners were filmed by a teacher doing an oral sex act.

The school is Vision Academy Nursery and Primary located in Muduuma sub county, Mpigi district. One of the investigators from the police told our reporter that when they arrived at the school, all teachers had run away, leaving behind only the security guard and the school bursar with about ten pupils, including the two victims who appeared in the widely shared video.

“It is a school with only five classes teaching up to primary three,"  the investigator said. "Officers have started taking statements from different learners and the adults found at the scene. We have been able to get the name of one of the teachers who filmed the learners; she has been only identified as teacher Juliet.”

More information obtained from Gerald Katongole, Mpigi district inspector of schools, indicates that the said institution opened up by someone only identified as Mulagira is not registered by the ministry of Education.

“The school is illegally operating," Katongole said during a telephone interview. "I had never heard of it and we can not even tell whether those who were teaching these children are really trained teachers.” 

URN has also learned that the school operates from a residential house which was rented to them by a family member after the passing on of the owner. The issue at hand came to light when Humphrey Tumwesigye, a concerned citizen and parent, wrote to the Uganda National Teachers Union (Unatu) seeking reprimand for adults, believed to be teachers, behind the filming of two pupils engaging in sexual acts.

In a letter dated October 12, 2022, Tumwesigye who didn’t put his name on the letter says he came across a video on social media showing pupils aged between 5-10 years being questioned by their teacher on camera. The video, depicts two pupils—a boy and a girl—responding to an adult female’s command to replicate what she had observed them doing. The female adult in the video was also wielding a stick giving signals that if they didn’t follow her instructions she was going to cane them.

“Repeat and do what you were doing exactly,” a female voice is heard ordering the pupils, who at first showed hesitation but subsequently, out of fear, accepted the instructions and so re-acted the sexual actions that they had been performing.

Although the video only shows two minors, information obtained by our reporter shows that there were two other pupils who, together with the two in the video, had been found by the teachers engaging in sexual acts.

Tumwesigye, who is a lawyer by profession, says that while he does not support the actions of the pupils, it was improper for a teacher or other adult in charge of looking after children to instruct the students to repeat the inappropriate behaviour, let alone record and circulate it on social media. Tom Matovu, a retired teacher, said that watching the widely-circulated video made him sick to the stomach. He questioned whether the adult in the video had teaching experience.

"We are doomed if the individual telling those kids to repeat what they had done is a teacher. Teachers are trained to know what is expected of children at different developmental stages and how to handle such cases,” Matovu noted. 

Matovu added that in their days, when such an incident occurred, only those involved would be handled without even making it known to the entire school, unless it was applicable.  

"With younger children, sexual experimentation, typically occurs between friends and has a game-like feel," he said. "When an adult sees it, typically, he or she is expected to talk to them to stop the behaviour, and the behaviour ends or decreases. Filming them creates more problems."

Elizabeth Kisakye, a child physiologist and teacher trainer, had not yet watched the video when our reporter approached. But she went into shock when a colleague in the office showed it to her. 

“Was this filmed in a school? That cannot be a teacher doing that,” Kisakye who also doubles as an education officer at the Education ministry commented. Many people who viewed the video have raised something similar to what she said in her remark.

Kisakye said by telling the learner to repeat what they were found doing, this was already damaging and stigmatizing. 

“While handling errant children one should not expose a child to negative reprisals – including additional physical or psychological harm, or to lifelong abuse, discrimination or rejection by their local communities; this is bad,” said Kisakye.

Filbert Baguma, the Unatu secretary general to whom the initial complaint was sent, says that if indeed those who filmed the pupils are teachers, they need serious re-tooling on how to handle and deal with children.

"You see in private schools sometimes, they handpick people and put them in charge of those young ones. The action that was done is unprofessional on the side of the teacher. Because even when you have found them in that act which is wrong, then you don't force them to repeat it so that you can see what they were doing. By the time you say repeat what you were doing, it means you have seen them, and there is no reason why you should force them to go back into the same. That is completely unprofessional and should not be heard of," said Baguma. 
          
Martin Kasagara Kiiza, the executive director National Children Authority, notes that besides the teachers' code of conduct, many laws in Uganda bar individuals from exploiting children.

“You cannot record and share pictures or videos of minors like that, it is not allowed in the law; the Children (Amendment) Act of 2016 outlaws this as a form of child exploitation,” Kiiza said, adding that of late many individuals have been seen recording and later sharing videos including children which he says should be stopped before the long arm of the law catches up with them.

Besides the Children (Amendment) act quoted by Kasagara, the Computer Misuse act also prohibits the distribution of material that depicts a child engaged in sexually suggestive or explicit conduct. A new amendment of the same act signed by the president last week also bars unauthorized access to data, and prohibits the sharing of data relating to children without authorization from their parents or guardians.

Meanwhile, Baguma and Kisakye say that although the teachers should be chastised for how they handled the situation, there is an underlying problem of child sexual immorality that needs to be addressed.

According to Baguma, sexual immorality has persisted in schools covertly for years, and policymakers, parents, and instructors have all been conspicuously mute on how to solve the problem. Previously, he continued, this was a regular occurrence among teenagers, but given additional circumstances of such youngsters being exposed to pornography through digital tools and media, this immorality is not only intensifying but also spreading to even younger children.

"But we need not to emotionally look at the teacher alone but we also need to look at the source of this behaviour, where the child is getting it from and then also cure it from there because you will send away the teacher but the child will continue to copy and implement with fellow learners," added Baguma. By  URN , The Observer