Novel Nucleic Acid Detection for Human Parvovirus B19 Based on Pyrococcus furiosus Argonaute Protein

Abstract

Parvovirus B19 (B19V) is pathogenic to humans and causes various human diseases. However, no antiviral agents or vaccines currently exist for the treatment or prevention of B19V infection. Therefore, developing sensitive and specific methods for B19V infection diagnosis is essential for accurate diagnoses. Previously, a Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-Cas12a (cpf1)-based electrochemical biosensor (E-CRISPR) with a picomole sensitivity for B19V detection was established. Herein, we set up a novel nucleic acid detection system based on Pyrococcus furiosus Argonaute (PfAgo)-mediated nucleic acid detection, targeting the nonstructural protein 1 (NS1) region of the B19V viral genome (abbreviated B19-NS1 PAND). Benefiting from independent protospacer adjacent motif (PAM) sequences, PfAgo can recognize their target with guide DNA (gDNA) that is easy to design and synthesize at a low cost. In contrast to E-CRISPR, without preamplification with Polymerase Chain Reaction (PCR), the Minimum Detectable Concentration (MDC) of three guide- or single guide-mediated B19-NS1 PAND was about 4 nM, approximately 6-fold more than E-CRISPR. However, when introducing an amplification step, the MDC can be dramatically decreased to the aM level (54 aM). In addition, the diagnostic results from clinical samples with B19-NS1 PAND revealed 100% consistency with PCR assays and subsequent Sanger sequencing tests, which may assist in molecular testing for clinical diagnosis and epidemiological investigations of B19V.

CRISPR/Cas and Argonaute-Based Biosensors for Pathogen Detection

“…The minimum detection concentration (MDC) for B19-NS1 PAND using either 3 wizards or single wizard is approximately 4 nM without PCR preamplification. This value is approximately 6 times higher than that of E-CRISPR …”

“…This value is approximately 6 times higher than that of E-CRISPR. 74 The utilization of the Argonaute protein offers a promising solution to the challenges faced by CRISPR/Cas systems. 72 Similar to the CRISPR/Cas system, they are both defense mechanisms of pathogens in nature to resist bacteriophage infection and other biological invasions, and belong to the microbial defense system MDS, which is a class of nucleic acidmediated restriction nucleic acid enzymes.…”

Pyrococcus furiosus Argonaute Based Detection Assays for Porcine Deltacoronavirus

“…In light of this evidence, novel PfAgo-based detection approaches have been developed. 19,20 Recombinase-aided amplification (RAA) technology and reverse transcription recombinase-aided amplification (RT-RAA) are to obtain recombinase from bacteria or fungi, and realize DNA or RNA amplification in vitro by cooperating with other enzymes. 21,22 In this work, RAA and RT-RAA were employed to amplify the nucleic acid before attempting PfAgo testing, since nucleic acid amplification is crucial for sensitivity in nucleic acid testing.…”

“…Finally, PfAgo is easier to store and carry since it remains active at high temperatures. In light of this evidence, novel PfAgo-based detection approaches have been developed. , …”

PfAgo-Based Zika Virus Detection

“…For ZIKV detection, the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can probe and distinguish ZIKV and four dengue virus (DENV) serotypes in infected human-patient bodily fluid samples at concentrations as low as one copy per microliter [ 22 ]. To avoid the high cost of guide RNA (gRNA) synthesis and alleviate the dependence of the protospacer-adjacent motif (PAM) in the CRISPR/Cas system, we recently developed a cost-effective Pf Ago-mediated nucleic acid detection (PAND) technology, which has been applied in different viral nucleic acid detections, including human papillomavirus 16 (HPV-16), severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), human parvovirus B19, and African swine fever virus (ASFV) [ 23 , 24 , 25 ]. During the procedure of PAND, three 5′-phosphorylated single-stranded guide DNAs (5’P-gDNAs) directed Pf Ago to cleave the ZIKV target DNA and thus spawn a 16nt 5′-phosphorylated single-stranded DNA (ssDNA), which, in turn, served as the new second-round gDNA bonded to the apo form of Pf Ago and incised its complementary molecular beacons, leading to the split of the quenchers from each other and subsequent fluorescence detection.…”