Rapid Detection of DNA and RNA Shrimp Viruses Using CRISPR-Based Diagnostics

Abstract

Timely detection of persistent and emerging pathogens is critical to controlling disease spread, particularly in high-density populations with increased contact between individuals and limited-to-no ability to quarantine. Standard molecular diagnostic tests for surveying pathogenic microbes have provided the sensitivity needed for early detection, but lag in time-to-result leading to delayed action. On-site diagnostics alleviate this lag, but current technologies are less sensitive and adaptable than lab-based molecular methods. Towards the development of improved on-site diagnostics, we demonstrated the adaptability of a loop-mediated isothermal amplification-CRISPR coupled technology for detecting DNA and RNA viruses that have greatly impacted shrimp populations worldwide; White Spot Syndrome Virus and Taura Syndrome Virus. Both CRISPR-based fluorescent assays we developed showed similar sensitivity and accuracy for viral detection and load quantification to real-time PCR. Additionally, both assays specifically targeted their respective virus with no false positives detected in animals infected with other common pathogens or in certified specific pathogen-free animals. IMPORTANCE The Pacific white shrimp (Penaeus vannamei) is one of the most valuable aquaculture species in the world but has suffered major economic losses from outbreaks of White Spot Syndrome Virus and Taura Syndrome Virus. Rapid detection of these viruses can improve aquaculture practices by enabling more timely action to be taken to combat disease outbreaks. Highly sensitive, specific, and robust CRISPR-based diagnostic assays such as those developed here have the potential to revolutionize disease management in agriculture and aquaculture helping to promote global food security.

Keywords: assay development; diagnostics; pathogens; rapid tests.

FIG 1

Schematic for CRISPR-based DNA and RNA detection in a one-pot reaction. When the TSV or WSSV target sequence is absent, there is no amplification and no CRISPR-Cas mediated cleavage of the reporter molecule. When the TSV or WSSV target sequence is present, there is isothermal amplification of the target sequence and simultaneous detection by CRISPR-Cas target binding and collateral cleavage of a fluorescent reporter. LAMP ± RT signifies the need for reverse transcriptase (RT) and LAMP for the amplification of the TSV RNA target or need for LAMP only for the amplification of the WSSV DNA target.

FIG 2

TSV and WSSV LAMP primer design and screening. (a) Annotated TSV consensus genome showing OIE standard diagnostic real-time PCR target region in black and LAMP target regions in teal, green, purple, and coral. (b) Annotated portions of WSSV consensus genome showing OIE standard diagnostic real-time PCR target region in black, the SHERLOCKv1 target region in brown, and LAMP target regions in blue, coral, gold, green, and magenta. (c) Amplification plot of triplicate LAMP reactions screening four different TSV primer sets (indicated by different colors) using 1,000,000 target copies of TSV. (d) Amplification plot of triplicate LAMP reactions screening five different WSSV primer sets (indicated by different colors) using 1,000,000 target copies of WSSV.

FIG 3

TSV and WSSV guide RNA screening. (a) Plots showing Cas12b collateral cleavage activity of each different TSV guide RNA when 1,000,000 (black) or 0 (gray) copies of TSV RNA target is present. (b) Plots showing Cas12b collateral cleavage activity of each different WSSV guide RNA when 400,000,000 (blue) or 0 (gray) copies of WSSV DNA target is present. “04g” is the same sequence as “04” but it was transcribed from a gBlock instead of an amplicon (see Materials and Methods for more details).

FIG 4

Sensitivity and specificity of SHERLOCK assays. SHERLOCK assay 10-fold standard curve showing Ct values for synthetic (a) TSV RNA and (b) WSSV DNA target with the dashed line indicating linear regression. Ct values (mean +/− s.d.); n.d., not detected from SHERLOCK specificity tests evaluating the detection of (c) TSV and (d) WSSV in shrimp samples known to be infected with TSV, WSSV, and other common white shrimp pathogens, including Vibrio spp. (causing EMS), EHP, IHHNV, IMNV, and pathogen-free shrimp (SPF). (+) and (-) indicate SHERLOCK results.

FIG 5

TSV SHERLOCK validation. (a) H & E-stained representative section from P. vannamei susceptible line after TSV exposure showing focal acute-phase infection in the gill characterized by spherical intracytoplasmic inclusions bodies (black arrows), pyknosis (red arrows) and karyorrhexis. (b) H & E-stained representative section from P. vannamei resistant line after TSV exposure showing no pathological signs characteristic of TSV infection. (c) TSV load quantified by qPCR in resistant (coral) and susceptible animals (teal). (d) Correlation between qPCR and TSV SHERLOCK quantification. Gray diagonal line indicates a 1:1 correlation.

FIG 6

Comparison of SHERLOCKv2 assays to existing molecular assays. (a) Magnitude fold change in viral copies estimated by SHERLOCKv2 assays relative to qPCR assays for samples tested in both qPCR and SHERLOCK methods. (b) Variation across technical replicates for WSSV samples tested in both SHERLOCKv1 and SHERLOCKv2 assays.

FIG 7

WSSV SHERLOCK assay validation. (a) Correlation between qPCR and WSSV SHERLOCKv2 assay quantification. (b) Correlation between WSSV SHERLOCKv2 and WSSV SHERLOCKv1 assays. Gray diagonal lines indicate a 1:1 correlation.