The development of rapid, sensitive, and user-friendly diagnostic methods for Chlamydia psittaci (C. psittaci) is critical for early diagnosis and effective control of infections. Current techniques, such as qPCR and next-generation sequencing, face limitations in accessibility and practicality for point-of-care testing (POCT). In this study, we present the WPTTS platform (one-step recombinase polymerase amplification (RPA)-CRISPR/Cas12b assay using weak protospacer adjacent motif (PAM) recognition with two-temperature shifting), which leverages the temperature-dependent PAM stringency of Alicyclobacillus acidophilus Cas12b (AapCas12b) to overcome the challenges of one-step CRISPR detection. By employing weak PAM sequences and a two-temperature protocol (37 °C for amplification, 60 °C for detection), WPTTS minimizes interference between RPA amplification and CRISPR cleavage, achieving a sensitivity of 102 copies/reaction—equivalent to two-step methods—while maintaining operational simplicity. The platform also demonstrates enhanced specificity, enabling single-base resolution at critical target positions. Clinical validation with 40 samples confirmed its superior performance, with 95

Monitoring viral loads in sewage can reflect the prevalence of infections within communities to a certain extent. Methods for concentrating and enriching viruses in sewage are also rapidly evolving. The magnetic bead method has been widely adopted for nucleic acid extraction due to its simplicity and high efficiency. In this study, we designed three pre-treatment procedures (T1: sedimentation for 30 min; T2: low-speed centrifugation at 2500×g for 5 min; T3: high-speed centrifugation at 8000×g for 5 min) to identify the optimal pre-treatment for enhancing viral nucleic acid concentration efficiency using the magnetic bead method. Spiked recovery tests were employed to compare the concentration efficiency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in sewage between the magnetic bead method and the traditional polyethylene glycol (PEG) precipitation method. Real sewage samples were further used to evaluate both methods for enriching enveloped viruses (SARS-CoV-2, influenza virus) and non-enveloped viruses (Norovirus, Rotavirus, Adenovirus). Results demonstrated that low-speed centrifugation (T2) served as the optimal pre-treatment for the magnetic bead-based concentration. The high-throughput magnetic bead method achieved significantly higher recovery rates for SARS-CoV-2, Norovirus, and Adenovirus compared to PEG precipitation. Thus, the high-throughput magnetic bead method can be widely applied for the concentration and concentration of diverse viruses in sewage.

Mycoplasma pneumonia (MP) is a common pathogen of human respiratory infections and one of the leading causes of community-acquired pneumonia. Early and rapid diagnosis of its infection is crucial for clinical treatment decisions. In this study, we innovatively combined recombinase polymerase amplification with Pyrococcus furiosus Argonaute protein (PfAgo) to establish a novel molecular diagnostic method for MP. This assay demonstrated high specificity with no cross-reactivity with other common respiratory pathogens. The limit of detection was 2 × 104 copies μl-1. Furthermore, evaluation using 37 clinical samples showed 100% specificity and 86.36% sensitivity compared to quantitative real-time polymerase chain reaction (qPCR). The entire workflow, including sample preparation, can be completed within 2.5 h and requires only basic instrumentation. This method holds great potential for application in primary healthcare settings and resource-limited regions.

In recent years, misdiagnosis or delayed diagnosis of Chlamydia psittaci (C. psittaci) infections has led to frequent outbreaks of severe public health events, such as severe pneumonia and respiratory distress, drawing increasing attention. Rapid and simple detection methods are vital for early intervention to reduce severity and mortality. In this study, we designed highly specific RPA primers and crRNA (CRISPR RNA) based on the highly conserved CPSIT_0429 gene in the C. psittaci genome, and preliminarily established a nucleic acid detection method for C. psittaci using the RPA-CRISPR/Cas12a system. In the two-step assay, the combination of the CPSIT_0429-F1/R1 primer pair and CPSIT_0429-crRNA2 achieved a detection limit of 2 × 10° copies/μL. Incorporating 20% glycerol enabled a one-tube assay with a limit of 2 × 102 copies/μL. Furthermore, the method showed no cross-reactivity with common respiratory pathogens such as influenza virus, SARS-CoV-2, and Streptococcus pneumoniae, demonstrating excellent specificity. Both the two-step and one-tube methods were compared with qPCR-verified C. psittaci positive samples. The results indicated that both assays showed high consistency with qPCR results. The RPA-CRISPR/Cas12a detection method is rapid, accurate, highly sensitive, and specific, providing a reliable platform for early diagnosis and clinical management of C. psittaci infections.

CRISPR/Cas12a is a highly promising detection tool. However, detecting single nucleotide variations (SNVs) remains challenging. Here, we elucidate Cas12a specificity through crRNA engineering and profiling of single- and double-base mismatch tolerance across three targets. Our findings indicate that Cas12a specificity depends on the number, type, location, and distance of mismatches within the R-loop. We also find that introducing a wobble base pair at position 14 of the R-loop does not affect the free energy change when the spacer length is truncated to 17 bp. Therefore, we develop a new universal specificity enhancement strategy via iterative crRNA design, involving truncated spacers and a wobble base pair at position 14 of the R-loop, which tremendously increases specificity without sacrificing sensitivity. Additionally, we construct a PAM-free one-pot detection platform for SARS-CoV-2 variants, which effectively distinguishes SNV targets across various GC contents. In summary, our work reveals new insights into the specificity mechanism of Cas12a and demonstrates significant potential for in vitro diagnostics. A new ultra-specific enhancement strategy for Cas12a via iterative crRNA design. A PAM-free one-pot detection platform for SARS-CoV-2 variants demonstrates significant potential for in vitro diagnostics.

Introduction. Chlamydia psittaci (C. psittaci) is a zoonotic infection, that causes psittacosis (parrot fever) in humans, leading to severe clinical manifestations, including severe pneumonia, adult respiratory distress syndrome, and, in rare cases, death.Gap Statement. Rapid, sensitive and specific detection of C. psittaci facilitates timely diagnosis and treatment of patients.Aim. This study aimed to engineer the LAMPCRISPR/Cas12b platform for C. psittaci detection.Methodology. The loop-mediated isothermal amplification (LAMP) technique and clustered regularly interspaced short palindromic repeats-CRISPR associated protein 12b (CRISPR-Cas12b) assay were combined to establish two -step and one -tube LAMPCRISPR/Cas12b reaction systems, respectively, for rapidly detecting C. psittaci. Results. The two -step and one -tube LAMPCRISPR/Cas12b assay could complete detection within 1 h. No cross-reactivity was observed from non -C. psittaci templates with specific LAMP amplification primers and single -guide RNA (sgRNA) targeting the highly conserved short fragment CPSIT_0429 gene of C. psittaci. The detection limits of the two -step and one -tube LAMPCRISPR/Cas12b reaction were 102 aM and 103 aM, respectively. The results were consistent with qPCR for nucleic acid detection in 160 clinical samples, including 80 suspected C. psittaci samples, kept in the laboratory.Conclusions. The LAMPCRISPR/Cas12b assay developed in this study provides a sensitive and specific method for rapidly detecting C. psittaci and offers technical support for its rapid diagnosis.

The occurrence of Giardia and Cryptosporidium (oo)cysts in drinking source water poses a serious public health risk. Here, we established a method that combines membrane concentration and real-time polymerase chain reaction (PCR) to quantify Giardia and Cryptosporidium in drinking water. The water samples were filtered through a cellulose membrane to collect Giardia and Cryptosporidium, and then nucleic acids were extracted. Specific primers and probes were designed and synthesized according to the gph gene sequence of Giardia and 18S rRNA gene sequence of Cryptosporidium. The concentrations of the two targets were determined using real-time PCR technology. The sensitivity, specificity, and stability of the method were evaluated. Our findings revealed that the detection limits of real-time PCR method for detecting Giardia and Cryptosporidium were 0.926 and 0.65 copy/µL, respectively; the spiked recovery rates were above 60% and 38%, respectively, and relative standard deviations were under 0.95% and 2.26%, respectively. Therefore, this effective procedure based on the membrane concentration method and real-time PCR will be useful for detecting Giardia and Cryptosporidium in drinking water for purpose of continuous environmental monitoring.

Norovirus is one of the major causes of outbreaks and sporadic cases of acute gastroenteritis in school children. Obtaining local genotype diversity information regarding norovirus is important for developing and evaluating prevention strategies of the transmission of this virus in school children. Clinical specimens, obtained from the routine acute gastroenteritis surveillance network from 2018 to 2019, were primarily tested using commercial real-time PCR Kit. Samples with Ct value less than 25 were selected and used for complete genome sequencing and those with Ct value between 25 and 30 were selected and used for he partial VP1 and RdRp regions sequencing. Phylogenetic trees of the viral genome were constructed by using the neighbor-joining method with bootstrap analysis of 1000 replicates in MEGA 6.0. Epidemiological surveillance of acute intestinal infections (n = 384) showed high-level detection (73.18%) of human norovirus in school endemic acute gastroenteritis events in Changzhou, with obvious epidemic characteristics in autumn and winter. Through genotyping, it was found that 93.12% of norovirus were GII, including GII.2, GII.3, GII.4, GII.6, GII.7, and GII.17. By October 2019, two norovirus genotypes, GII.4[P31] and GII.17[P17], became the preponderant epidemic strains. Phylogenetic analysis of the new GII.17[P17] complete genomes showed close relationship with Miyagi strain identified in Japan in 2015, and GII.4[P31] showed close relationship with Jinan strain indentified in China in 2017. The study highlights the emerging role of GII.4[P31] and GII.17[P17] in causing endemic acute gastroenteritis outbreaks at school children, in Changzhou, China in 2019.

ABSTRACT The full-length genome sequence of a human enterovirus 71 (EV71) strain (EV71/CZTN01/CHN/2017) was isolated from a throat swab from a child in Changzhou, China, in 2017. According to the phylogenetic analyses, the full-genome sequence in this study belongs to sub-subgenotype C4a.

Norovirus is the leading global cause of epidemic gastroenteritis and responsible for more than 90% of all viral gastroenteritis as well as 50% of gastroenteritis outbreaks worldwide. During February-March 2017, a sharp increase in the number of norovirus outbreaks was reported by the Changzhou Center for Disease Control and Prevention in China. A total of 250 anal swabs were collected from 24 outbreaks, and the genotypes were determined by sequencing analysis. The genetic diversity, epidemiological status, and characteristics of the norovirus strains were analyzed. A novel recombinant GII.P16-GII.2 norovirus strain was identified as the primary cause of these outbreaks. Phylogenetic trees showed that the novel GII.P16-GII.2 strains were similar to the GII.P16-GII.4 strains (GenBank accession nos. KX907727.1, USA, 2015; LC175468.1, Japan, 2016) for RdRp gene. It also showed similarity to the GII.P16-GII.2 recombinants (GenBank accession no. LC145787.1, Japan, 2012; KJ407074.2, USA, 2011) for VP1 gene. Thus, the novel GII.P16-GII.2 recombinants might have evolved from GII.P16-GII.4 strains in 2015-2016 and GII.P16-GII.2 in 2011-2012.

The aim of this study was to analyze the genetic characteristics of the VP1 gene of coxsackievirus A16(CA16)strains isolated from Jiangsu Province, China, in 2015.The VP1 regions of 20CA16 virus strains from Jiangsu Province in 2015 were amplified, and the amplification products were sequenced. Mega 6.0and DNA Star software were applied to build the phylogenetic tree and analyze the homogeneity of nucleotides and amino acids. The sequence homologies of the nucleotides and amino acids of the VP1 gene were 88.2%~100.0%and 98.0%~100.0%among 20CA16 isolates, respectively. Comparison with the sequence of the prototype strain A-G10 showed 75.3%~77.4% homologies in nucleotide sequence and90.6%~92.3%in amino-acid sequences, respectively. Comparison with the sequence of the representative strain B1 showed 88.3%~98.4% homologies in nucleotide sequence and 96.3%~100.0%in amino-acid sequence, respectively. Comparison with the sequence of the representative strain B2 showed 88.4%~90.8% homologies in nucleotide sequence and 96.6%~100.0%in amino-acid sequence, respectively. Twenty CA16 isolates were subgenotype B1.One isolate was subgenotype B1a, whereas the remainder of isolates was subgenotype B1b among 20 CA16 isolates. A subgenotype B1b transmission chain was also noted. The CA16 strains isolated from Jiangsu Province in 2015 belonged to subgenotype B1.There were two evolutionary branches, whereby B1 a and B1bwere co-circulating and evolving together. The epidemic strain was subgenotype B1b.