CRISPR-associated (Cas) protein systems have been increasingly incorporated in nucleic-acid diagnosis. CRISPR/ Cas12a can cleave single-stranded DNA (ssDNA) after being guided to the target double-stranded DNA (dsDNA) with crRNA, making it a specific tool for dsDNA detection. Assisted by nucleic acid preamplification, CRISPR/Cas12a enables dsDNA detection at the attomolar level. However, such mandatory preamplification in CRISPR/Cas12a also accompanies the extra step of transferring preamplification products into the CRISPR/Cas12a system, which is not only cumbersome and time-consuming but also induces the risk of cross-contamination. Herein, we demonstrate a multiplex-crRNA strategy to enhance the sensitivity of the CRISPR/Cas12a system without any preamplification. This multiplex-crRNA strategy harnesses multiple sequences of crRNA which target different regions of the same dsDNA substrate in the same CRISPR/Cas12a system. Therefore, detection signals are accumulated without amplification, which augments the conventional detection limit. For application demonstration, the B646L gene from the African swine fever virus (ASFV), which is a dsDNA virus, is exemplified. The detection limit of the multiplex-crRNA system can be improved to ∼1 picomolar (pM) without amplification, which is ∼64 times stronger than the conventional single-crRNA system. The multiplex-crRNA system presented in this study, with slight modifications, can be generalized to other biosensing settings where preamplification is not readily available.

Pulse-Position Modulation (PPM) is a modulation method that only makes every pulse in the carrier pulse sequence change with time but without changing the shape and amplitude of the pulse signal. In this paper, a PPM system is designed. Firstly, an appropriate mathematical model is established to represent PPM transmission, and the shape of the pulse signal is designed. After that, we write the code and add a white Gaussian noise channel. Then the transmission process is simulated and visualized. At last, the error rate of the scheme is analyzed and discussed through MATLAB simulation then compared with other modulation methods. The goal of this paper is to study PPM by designing a PPM system fully. Besides, our method is compared with other modulation methods to understand the advantages and disadvantages of PPM. This may help other scholars to design and research the PPM system.

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