The mutation status of epidermal growth factor receptor (EGFR) exon 19 is of great importance for predicting sensitivity to tyrosine kinase inhibitors (TKIs) in the treatment of non-small-cell lung cancer (NSCLC). However, the development of simple, sensitive, and no-nonspecific amplification platforms for EGFR 19del detection in NSCLC remains a challenge. Herein, we developed a novel, simple, and highly sensitive naked-eye assay utilizing CRISPR/Cas12a-triggered no-nonspecific nucleic acid amplification (NAA) with rolling circle amplification (RCA) as a model for EGFR 19del detection. Typically, circular padlocks are designed to be the trans-cleavage substrate of Cas12a/crRNA and serve as templates for RCA. Since the target EGFR 19del induces robust trans-cleavage activity of the Cas12a/crRNA duplex, the surrounding circular padlocks are cleaved into random short linear fragments that are unable to initiate RCA, resulting in a colorless solution. However, in the absence of EGFR 19del, the inactivated Cas12a enzymes cannot cleave the circular padlocks, and they remain able to serve as templates to initiate RCA to generate long single-stranded DNA to further fold into G-quadruplex/hemin DNAzymes to catalyze the oxidation of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS 2− ), generating a color response that is obvious to the naked eye. As expected, this strategy with a detection limit as low as 20 fM exhibited robust selectivity and anti-interference ability. Moreover, this method was applicable for detecting EGFR 19del in real serum samples and showed high consistency with real-time quantitative polymerase chain reaction (qPCR) and sequencing results, providing a promising strategy for the early noninvasive diagnosis and guidance of clinical treatment for cancer.

Previous research indicates that the structural gene coding for the neurotoxin is on a plasmid [1,2]. The neurotoxin can be endocytosed by both Rab5-and clathrin-dependent mechanisms and then transported to the central nervous system (CNS) by long-distance retrograde axonal transport, where it can inhibit the release of neurotransmitters in the spinal cord, leading to periodic hyper contraction of skeletal muscles (termed spastic paralysis) [3]. The spores of C. tetani are abundantly found in soil and other environments; in most cases, tetanus originates from damaged skin or narrow wounds, such as those resulting from dental infections, intravenous drug abuse, compound fractures, or nail punctures. These areas confer a more favorable anaerobic environment for the growth and reproduction of C. tetani [4,5]. Tetanus can be prevented by active immunization of children and pregnant women. There is no effective treatment for this disease. Tetanus management involves aggressive airway management, treatments to inhibit the absorption of toxins, strict sanitary measures, treatments to alleviate muscle spasms, high-intensity therapy, and intensive supportive care [6,7]. Tetanus generally occurs in low and middle-income countries but has a low incidence in developed countries [6,8]. Maternal and neonatal tetanus (MNT) maintains a widespread public health issue, with mortality between 80% and 100% among neonates [9]. It is estimated that one million cases of tetanus happen worldwide annually and led to between 48199 and 80042 deaths in 2015. The World Health Organization (WHO) estimated that 30,848 newborns died of neonatal tetanus in 2017, and the imputed number of neonatal tetanus deaths decreased to 25000 in 2018 [10,11]. Although mortality is steadily declining, we believe that the global incidence of the disease is underestimated because most cases arise in resource-limited settings where surveillance systems are limited, and accurate data cannot be obtained. Over the past few decades, intensive care measures have undergone significant changes in numerous countries, and it is possible that high tetanus incidence rates persist despite the remarkable declines in mortality that have occurred [12][13][14]. This is an additional factor that may result in an underestimation of disease.The diagnosis of tetanus typically relies on the clinical history and major symptoms. In localized tetanus, differential diagnosis is necessary, and laboratory tests can exclude any other diseases with similarities to tetanus, such as strychnine poisoning, neuroleptic malignant syndrome, stiff-person syndrome, and dystonic reaction to Tetanus is a potentially fatal public health illness resulted from the neurotoxins generated by Clostridium tetani. C. tetani is not easily culturable and culturing the relevant bacteria from infected wounds has rarely been useful in diagnosis; PCR-based assays can only be conducted at highly sophisticated laboratories. Therefore, a real-time recombinase polymerase amplification assay (Exo-RPA) was constructed to ...

Lung cancer is the highest incidence of malignant tumors in the world. Targeted therapy based on gene mutation detection including epidermal growth factor receptor (EGFR) gene mutation is one of the first-line treatments. In this work, took EGFR gene G719S mutation as a representative object, a large number hexagonal cavities microfluidic chip (LHMC) platform based on digital PCR (dPCR) for the absolute quantification of gene mutation in lung cancer was established. In this chip, 113,137 cavities chip was designed to increase the number of absolute quantitative, which was 2~5 times higher than the traditional one. The hexagonal shape of cavities elevates the filling rate and filling speed. A set of primers and probes for G719S with high sensitivity, high specificity and high reliability were designed and screened. Then the PCR parameters were optimized. The results demonstrated that the chip platform shows good performance. The minimum detection concentration of the gene mutant was 3.01 copies/μL, and a good correlation (Y= 0.725X- 0.581, R2= 0.984) was noted between the measured value and the expected value. This chip possesses sensitively detect positive mutations in G719S and completely negative when detecting other substances. The developed LHMC-dPCR chip is a rapid and accurate gene mutation analysis platform, which has faster speed and lower price than classic detection methods, for instance, droplet dPCR, DNA sequencing method. Moreover, LHMC-dPCR is not limited by the number of nucleic acids and droplets and there is no need to estimate the nucleic acid concentration of the sample. This chip platform could also detect other gene mutations, for example, L858R, exon 19 deletions, in other tumors including lung cancer.