Up to now many UDG assays have been reported based

Up to now, many UDG assays have been reported based on the design of dU-containing substrate DNA, which can be roughly divided into two categories according to the sensing mechanisms. One type of UDG assays are simply based on the physical or chemical property changes of the substrate accompanied with the removal of dU without involvement of nucleic Epidermal Growth Factor (EGF), human recombinant sale amplification strategies [13], [14], [15], [16], [17]. These methods are simple, but they suffer from relatively low sensitivity due to the lack of signal amplification routes. Based on the different functional DNA structures, the other kind of UDG assays generally combine with the various nucleic acid amplification strategies in order to achieve higher sensitivity [18], [19], [20], [21], [22], [23]. Techniques such as loop-mediated isothermal amplification (LAMP) [21], rolling circle amplification (RCA) [22], and isothermal exponential amplification reaction (EXPAR) [23] are widely used due to their superior sensitivities. Although these methods are ingenious, most of them still involve tedious manipulations and sophisticated DNA design. Therefore, the development of simple but highly sensitive UDG assays is still urgently desired. Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase that can add dNTPs to the 3′-OH ends of DNA without requirement of any template. Previous studies have demonstrated that dTTP showed the greatest extension capacity among the four nucleotides under the catalysis of TdT [24]. Herein, we wish to propose a TdT-assisted highly sensitive method to detect UDG activity. Firstly, a 3′-ddC (2′, 3′-dideoxycytosine)-blocked hairpin DNA substrate is rationally designed which ensures extremely low nonspecific background than those of other 3′-end blocking moieties. UDG can trigger the BER mechanism by removing the uracil base from the substrate DNA and then endonuclease IV (Endo IV) will split off the DNA into short fragments bearing free 3′-OH. Afterward, TdT catalyzes the template-free DNA extension along such 3′-OH fragments to form long poly(T) sequences. Finally, these elongated poly(T) sequences can serve as ideal templates to form intensely fluorescent copper nanoclusters (CuNCs) based on the sodium ascorbate-assisted reduction of Cu2+ [25], [26], [27], [28]. Each poly(T) with thousands of thymines can form many CuNCs emitting strong red fluorescence at 650 nm, which greatly amplifies the fluorescence response for the sensitive detection of UDG activity. It is worth noting that unlike the widely investigated biomolecules-templated AuNCs [29], [30] and AgNCs [31], [32], the preparation of which both require quite long time and high concentrations of templates (e. g., >µM DNA), the formation of CuNCs only needs less DNA templates in the nM scale and can be completed within several minutes with more simplified and controllable procedures. Moreover, with the assistance of an AP-site-containing poly(A) oligonucleotide, we have further devised a hyperbranched TdT-extension mechanism. The lowest detectable of UDG can be further pushed down to an extremely low level of 0.000002 U/mL, which is one of the most lower detection limits for the detection of UDG.
Experimental
Results and discussion
Conclusion
Acknowledgements This work was supported by the National Natural Science Foundation of China (21622507, 21575086), Program for Changjiang Scholars and Innovative Research Team in University, China (IRT 15_R43), and the Fundamental Research Funds for the Central Universities (GK201802016).
Introduction Corynebacterium pseudotuberculosis, a Gram-positive bacterium, infects a variety of hosts, including humans, but predominantly affects sheep and goats, in which it is the causal agent of caseous lymphadenitis (Dorella et al., 2006, Seyffert et al., 2014). Found worldwide, this disease is responsible for substantial economic losses in small ruminant production, by decreasing meat, milk, and wool yields, reducing breeding capacity, and rendering hides and even carcasses of slaughtered animals unfit for use (Jeber et al., 2016; Soares et al., 2013). In the host organism, C. pseudotuberculosis survives within phagocytes, inside which it is carried to lymph nodes, where the formation of characteristic abscesses follows an inflammatory period (Stefańska et al., 2010, Windsor, 2011).