Integrating CRISPR/Cas within isothermal amplification for point-of-Care Assay of nucleic acid.

Pathogen detection strategy based on CRISPR

Emerging and relapsing infectious diseases pose a huge health threat to human health and a new challenge to global public health. Rapid, sensitive and simple diagnostic tools are keys to successful management of infectious patients and containment of disease transmission. In recent years, international research on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-related proteins (Cas) has revolutionized our understanding of biology. The CRISPR-Cas system has the advantages of high specificity, high sensitivity, simple, rapid, low cost, and has begun to be used for molecular diagnosis and treatment of infectious diseases. In this paper, we described the biological principles, application fields and prospects of CRISPR-Cas system in the molecular diagnosis and treatment of infectious diseases, and compared it with existing molecular diagnosis methods, the advantages and disadvantages were summarized. 

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The CRISPR-Cas system as a tool for diagnosing and treating infectious diseases

COVID-19 was officially declared a global pandemic disease on 11 March 2020, with severe implications for healthcare systems, economic activity, and human life worldwide. Fast and sensitive amplification of the severe acute respiratory syndrome virus 2 (SARS-CoV-2) nucleic acids is critical for controlling the spread of this disease. Here, a real-time reverse transcription recombinase-aided amplification (RT-RAA) assay, targeting conserved positions in the nucleocapsid protein gene (N gene) of SARS-CoV-2, was successfully established for SARS-CoV-2. The assay was specific to SARS-CoV-2, and there was no cross-reaction with other important viruses. The sensitivity of the real-time RT-RAA assay was 142 copies per reaction at 95% probability. Furthermore, 100% concordance between the real-time RT-RAA and RT-qPCR assays was achieved after testing 72 clinical specimens. Further linear regression analysis indicated a significant correlation between the real-time RT-RAA and RT-qPCR assays with an R2 value of 0.8149 (p < 0.0001). In addition, the amplicons of the real-time RT-RAA assay could be directly visualized by a portable blue light instrument, making it suitable for the rapid amplification of SARS-CoV-2 in resource-limited settings. Therefore, the real-time RT-RAA method allows the specific, sensitive, simple, rapid, and reliable detection of SARS-CoV-2.

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Real-Time Reverse Transcription Recombinase-Aided Amplification Assay for Rapid Amplification of the N Gene of SARS-CoV-2

A point-of-care rapid HIV-1 test using an isothermal recombinase-aided amplification and CRISPR Cas12a-mediated detection

A longitudinal serological study to investigate the seropositive frequency, incidence, and antibody dynamics of Chlamydia trachomatis infection in the general population of China is urgently needed in order to optimize the strategies for surveillance and precise prevention of C. trachomatis infection. This longitudinal study enrolled 744 subjects aged 18-65 years from Jidong Community of Northern China from 2014 to 2018. Seropositive frequency, incidence, and reinfection of C. trachomatis were determined by detecting antibody against C. trachomatis Pgp3 using "in-house" luciferase immunosorbent assay (LISA). The dynamic of anti-Pgp3 antibody was analyzed using the Generalized Estimating Equation (GEE) model. The overall Pgp3 seropositive frequency among the 18-65-year-old population was 28.1% (95% CI 24.9-31.5), and significantly increased from 12.0% in those aged 18-29 years to 48.6% in the 60-65 years old. The seropositive frequency was slightly higher in women than in men (31.3% vs. 25.4%) without statistical significance. The C. trachomatis incidence and reinfection rate were 11 and 14 per 1,000 person-years, respectively, and showed no significant difference with respect to age, gender, ethnicity, marital status, and education levels. Furthermore, anti-Pgp3 antibody remained detectable in 93.3% (195/209) of the seropositive subjects during the 5 years of follow-up. The overall decay rate for anti-Pgp3 antibody for CT-infected persons was -0.123 Log2 RLU/year, which was dramatically slower than in CT new infection (-3.34 Log2 RLU/year) or reinfection (-1.1 Log2 RLU/year). In conclusion, at least one quarter of the people aged 18-65 years have been infected with C. trachomatis over their lifetime while all age groups are susceptible to C. trachomatis infection in the community of Northern China. Therefore, comprehensive prevention strategies are urgently needed.

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Seroepidemiology of Chlamydia trachomatis Infection in the General Population of Northern China: The Jidong Community Cohort Study.

A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type (n = 5) and alpha (n = 11), beta (n = 8), and delta variants (n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.

Hongqing Lin

Papers

A point-of-care rapid HIV-1 test using an isothermal recombinase-aided amplification and CRISPR Cas12a-mediated detection

Seroepidemiology of Chlamydia trachomatis Infection in the General Population of Northern China: The Jidong Community Cohort Study.

CRISPR-Cas12a-Based Detection for the Major SARS-CoV-2 Variants of Concern.