One-Pot Visual Detection of African Swine Fever Virus Using CRISPR-Cas12a

African swine fever virus (ASFV) is a leading cause of worldwide agricultural loss. ASFV is a highly contagious and lethal disease for both domestic and wild pigs, which has brought enormous economic losses to a number of countries. Conventional methods, such as general polymerase chain reaction and isothermal amplification, are time-consuming, instrument-dependent, and unsatisfactorily accurate. Therefore, rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we created a one-pot visual detection system for ASFV with CRISPR/Cas12a technology combined with LAMP or RPA. A mineral oil sealing strategy was adopted to mitigate sample cross-contamination between parallel vials during high-throughput testing. Furthermore, the blue fluorescence signal produced by ssDNA reporter could be observed by the naked eye without any dedicated instrument. For CRISPR-RPA system, detection could be completed within 40 min with advantageous sensitivity. While CRISPR-LAMP system could complete it within 60 min with a high sensitivity of 5.8 × 102 copies/μl. Furthermore, we verified such detection platforms display no cross-reactivity with other porcine DNA or RNA viruses. Both CRISPR-RPA and CRISPR-LAMP systems permit highly rapid, sensitive, specific, and low-cost Cas12a-mediated visual diagnostic of ASFV for point-of-care testing (POCT) applications

Small extrachromosomal circular DNAs as biomarkers for multi‐cancer diagnosis and monitoring

Abstract Background Small extrachromosomal circular DNAs (eccDNAs) have the potential to be cancer biomarkers. However, the formation mechanisms and functions of small eccDNAs selected in carcinogenesis are not clear, and whether the small eccDNA profile in the plasma of cancer patients represents that in cancer tissues remains to be elucidated. Methods A novel sequencing workflow based on the nanopore sequencing platform was used to sequence naturally existing full‐length small eccDNAs in tissues and plasma collected from 25 cancer patients (including prostate cancer, hepatocellular carcinoma and colorectal cancer), and from an independent validation cohort (including 7 cancer plasma and 14 healthy plasma). Results Compared with those in non‐cancer tissues, small eccDNAs detected in cancer tissues had a significantly larger number and size (P = 0.040 and 2.2e‐16, respectively), along with more even distribution and different formation mechanisms. Although small eccDNAs had different general characteristics and genomic annotation between cancer tissues and the paired plasma, they had similar formation mechanisms and cancer‐related functions. Small eccDNAs originated from some specific genes had great multi‐cancer diagnostic value in tissues (AUC ≥ 0.8) and plasma (AUC > 0.9), especially increasing the accuracy of multi‐cancer prediction of CEA/CA19‐9 levels. The high multi‐cancer diagnostic value of small eccDNAs originated from ALK&ETV6 could be extrapolated from tissues (AUC = 0.804) to plasma and showed high positive predictive value (100%) and negative predictive value (82.35%) in a validation cohort. Conclusions As independent and stable circular DNA molecules, small eccDNAs in both tissues and plasma can be used as ideal biomarkers for cost‐effective multi‐cancer diagnosis and monitoring