Completely Free from PAM Limitations: Asymmetric RPA with CRISPR/Cas12a for Nucleic Acid Assays.

Experimentally, Cas12a can recognize multiple protospacer adjacent motif (PAM) sequences and is not restricted to the "TTTN". However, the application of the CRISPR/Cas12a system is still limited by the PAM for double-stranded DNA (dsDNA). Here, we developed asymmetric RPA (Asy-RPA) to completely break the limitations of PAM. Asy-RPA not only achieved efficient amplification but also converted dsDNA to single-stranded DNA (ssDNA) without complicated steps. The ssDNA products activated the trans-cleavage activity of Cas12a, outputting signals. The application of Asy-RPA completely freed Cas12a from the PAM, which can be more widely used in nucleic acid detection, such as lumpy skin disease virus, with an actual detection limit as low as 1.21 × 101 copies·µL-1. More importantly, Cas12a was intolerant to mutations on ssDNA. This provided technical support for the detection and identification of wild-type Mycobacterium tuberculosis (WT-TB) and rifampin-resistant mutant-type M. tuberculosis (MT-TB). The detection limit was as low as 1 fM for 1% mixed samples. The detection and availability of different treatment options for treatment-resistant and WT-TB were significant for the elimination of TB. In summary, the platform consisting of Asy-RPA and CRISPR/Cas12a was suitable for the detection of various viruses and bacteria and was a boon for the detection of dsDNA without recognizable PAM.

Field-friendly and ultra-fast detection platform without nucleic acid extraction for virus detection.

The polymeric chain reaction (PCR) has come under fire for being time-consuming, requiring expensive equipments, and requiring the extraction and purification of nucleic acids. Here, an ultra-fast and sensitive detection platform without nucleic acid extraction solved the above problems. Firstly, the RoomTemp Sample Lysis Kit released the nucleic acid in 3 min and removed the inhibition to facilitate the amplification reaction. What's more, ultra-fast PCR (UF-PCR) can complete 40 cycles in just 15 min and 50 s. To improve the sensitivity and provide more convenient reading modes, CRISPR/Cas12a was mediated to detect Lumpy skin disease virus (LSDV). The platform output fluorescence and Lateral flow dipstick (LFD) signals. The actual detection limit was 2 × 101 copies·µL-1. The portable platform realized visualization, excellent sensitivity and quick speed. In summary, the field-friendly testing platform had great potential in practical testing.

Enzyme-free and sensitive method for single-stranded nucleic acid detection based on CHA and HCR.

Simple, rapid, and highly sensitive methods for single-stranded nucleic acid detection are of great significance in clinical testing. Meanwhile, common methods are inseparable from the participation of enzymes, which greatly increases their complexity. Herein, an enzyme-free and sensitive method combining HCR and CHA is established to detect single-stranded nucleic acid. A target induces the auxiliary hairpin strands to open their secondary structure, exposing partial sequences that can trigger catalytic hairpin assembly (CHA) and hybridization chain reactions (HCR), respectively. To avoid additional signaling substances, 2-aminopurines (which fluoresces differently in double-stranded DNA and G-quadruplex) are modified in the substrate chains of CHA and HCR. Compared with methods that adopt CHA or HCR alone, the sensitivity of this method is increased by nearly 10 times. Moreover, this method can effectively improve the specific recognition of the target. To "turn on" the method, two regions that can pair with H5 and H6 are required. Taking foot-and-mouth disease virus (FMDV) as the object, this method can specifically detect FMDV to 2.78 × 101 TCID50. Although the sensitivity is not as good as RT-qPCR, it owns the advantages of simplicity and speed. We think this method can be used for the primary screening of FMDV, and has application potential in some grassroots.

Development of the isothermal recombinase polymerase amplification assays for rapid detection of the genus Capripoxvirus.

Sheeppox virus (SPPV), goatpox virus (GTPV) and lumpy skin disease virus (LSDV) belong to the genus Capripoxvirus (CaPV), and are important pathogens of sheep, goat and cattle, respectively. Rapid and reliable detection of CaPV is critical to prevent its spread and promote its eradication. This study aimed to develop the recombinase polymerase amplification (RPA) assays combined with real-time fluorescence (real-time RPA) and naked-eye visible lateral flow strip (LFS RPA) for rapid detection of CaPV. Both developed RPA assays worked well at 39 °C within 20 min. They were highly specific for the detection of GTPV, SPPV and LSDV, while no cross-reactivity was observed for other non-targeted pathogens and genomic DNA of goat, sheep and cattle. The limit of detection for real-time RPA and LFS RPA were 1.0 × 102 and 1.0 × 101 copies per reaction, respectively. In the artificially contaminated samples with GTPV, the detection results of RPA assays were consistent with those of real-time PCR. For 15 clinical samples, LSDV was detected by real-time RPA, LFS RPA and real-time PCR in 13, 15 and 15 samples, respectively. The developed RPA assays were specific, sensitive, and user-friendly for the rapid detection of CaPV, and could be a better alternative method applied in low-resources settings.

The End of the Gray Zone: One-Tube Nested Recombinase Polymerase Amplification with Ultrahigh Signal-to-Noise Ratio for Precisely Detecting and Surveilling Viruses.

The samples were difficult to accurately determine positive or negative between 35 and 40 cycles by real-time quantitative PCR (qPCR) as the standard method. Here, we developed one-tube nested recombinase polymerase amplification (ONRPA) technology with CRISPR/Cas12a to overcome this difficulty. ONRPA broke the amplification plateau to substantially enhance the signals, which considerably improved the sensitivity and eliminated the problem of gray area. Using two pairs of primers one after another, it improved precision by lowering the probability of magnifying several target zones, which was completely free of contamination by nonspecific amplification. This was important in nucleic acid testing. Finally, by the CRISPR/Cas12a system as a terminal output, the approach achieved a high signal output as few as 2.169 copies·µL-1 in 32 min. ONRPA was 100-fold more sensitive than conventional RPA and 1000-fold compared to qPCR. ONRPA coupled with CRISPR/Cas12a will be an important and new promoter of RPA in clinical applications.

Current Knowledge on Epizootic Haemorrhagic Disease in China.

Epizootic haemorrhagic disease (EHD) is an infectious, non-contagious viral disease of ruminants caused by epizootic haemorrhagic disease virus (EHDV) and is transmitted by insects of the genus Culicoides. In 2008, EHD was listed on the World Organization for Animal Health (WOAH) list of notifiable terrestrial and aquatic animal diseases. This article reviews the distribution of EHD in China and relevant studies and proposes several suggestions for the prevention and control of EHD. There have been reports of positivity for serum antibodies against EHDV-1, EHDV-2, EHDV-5, EHDV-6, EHDV-7, EHDV-8 and EHDV-10 in China. Strains of EHDV-1, -5, -6, -7, -8 and -10 have been isolated, among which the Seg-2, Seg-3 and Seg-6 sequences of serotypes -5, -6, -7 and -10 belong to the eastern topotype. The emergence of western topotype Seg-2 in EHDV-1 strains indicates that EHDV-1 strains in China are reassortant strains of the western and eastern topotypes. A novel serotype strain of EHDV named YNDH/V079/2018 was isolated in 2018. Chinese scholars have successfully expressed the EHDV VP7 protein and developed a variety of ELISA detection methods, including antigen capture ELISA and competitive ELISA. A variety of EHDV nucleic acid detection methods, including RT-PCR and qRT-PCR, have also been developed. LAMP and the liquid chip detection technique are also available. To prevent and control EHD, several suggestions for controlling EHD transmission have been proposed based on the actual situation in China, including controlling the number of Culicoides, reducing contact between Culicoides and hosts, continued monitoring of EHDV and Culicoides in different areas of China and further development and application of basic and pioneering research related to EHD prevention and control.

Multiple accurate and sensitive arrays for Capripoxvirus (CaPV) differentiation.

Capripoxvirus (CaPV) contains three viruses that have caused massive losses in the livestock and dairy industries. Accurate CaPV differentiation has far-reaching implications for effectively controlling outbreaks. However, it has a great challenge to distinguishing three viruses due to high homology of 97%. Here, we established a sensitive CRISPR/Cas12a array based on Multiple-recombinase polymerase amplification (M-RPA) for CaPV differentiation, which provided a more comprehensive and accurate differentiation mode targeting VARV B22R and RPO30 genes. By sensitive CRISPR/Cas12a and M-RPA, the actual detection limits of three viruses were as low as 50, 40 and 60 copies, respectively. Moreover, Lateral flow dipstick (LFD) array based on CRISPR/Cas12a achieved portable and intuitive detection, making it suitable for point-of-care testing. Therefore, CRISPR/Cas12a array and LFD array paved the way for CaPV differentiation in practice. Additionally, we constructed a real-time quantitative PCR (qPCR) array to fill the qPCR technical gap in differentiation and to facilitate the quarantine departments.

Carbon nanodots combined with loop-mediated isothermal amplification (LAMP) for detection of African swine fever virus (ASFV).

The spread of African swine fever virus (ASFV) caused huge economic costs, so early detection is particularly important. Here, we established a fluorescence biosensor based on carbon nanodots (CNDs) and loop-mediated isothermal amplification (LAMP) to ultra-sensitively detect ASFV. LAMP with high efficiency produced a large amount of pyro phosphoric acid and caused pH change in a short time. CNDs with strong light stability had a large fluorescence response at the emission wavelength of 585.5 nm to small pH change by the excitation wavelength of 550 nm. The biosensor realized "turn-off-on" mode for ASFV detection with the detection limit as low as 15.21 copies µL-1. In addition, the biosensor had high accuracy in the actual sample assay. Therefore, the biosensor achieved rapid, sensitive, low-cost, and simple detection for ASFV. Moreover, the biosensor broadened the detection pathway of LAMP as a tool with great development prospect.

One-pot platform for rapid detecting virus utilizing recombinase polymerase amplification and CRISPR/Cas12a.

The livestock industry has been deeply affected by African swine fever virus (ASFV) and Capripoxvirus (CaPV), which caused an enormous economic damage. It is emergent to develop a reliable detection method. Here, we developed a rapid, ultra-sensitive, and one-pot DNA detection method combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a for ASFV and CaPV, named one-pot-RPA-Cas12a (OpRCas) platform. It had the virtue of both RPA and CRISPR/Cas12a, such as high amplification efficiency, constant temperature reaction, and strict target selectivity, which made diagnosis simplified, accurate and easy to be operated without expensive equipment. Meanwhile, the reagents of RPA and CRISPR/Cas12a were added to the lid and bottom of tube in one go, which overcame the incompatibility of two reactions and aerosol contamination. To save cost, we only need a quarter of the amount of regular RPA per reaction which is enough to achieve clinical diagnosis. The OpRCas platform was 10 to 100 times more sensitive than qPCR; the limit of detection (LOD) was as low as 1.2 × 10-6 ng/µL (3.07 copies/µL by ddPCR) of ASFV and 7.7 × 10-5 ng/µL (1.02 copies/µL by ddPCR) of CaPV with the portable fluorometer in 40 min. In addition, the OpRCas platform combined with the lateral flow assay (LFA) strip to suit for point-of-care (POC) testing. It showed 93.3% consistency with qPCR for clinical sample analysis. Results prove that OpRCas platform is an easy-handling, ultra-sensitive, and rapid to achieve ASFV and CaPV POC testing. KEY POINTS: • The platform realizes one-pot reaction of RPA and Cas12a. • Sensitivity is 100 times more than qPCR. • Three output modes are suitable to be used to quantitative test or POC testing.

Non-nucleic acid extraction and ultra-sensitive detection of African swine fever virus via CRISPR/Cas12a.

Early diagnosis of the African swine fever virus (ASFV) is the main preventive measure for ASFV. Here, we developed a fluorescent biosensor and lateral flow assay (LFA) strip based on direct PCR combined with CRISPR/Cas12a system for ASF. Direct PCR can simultaneously split samples and efficiently amplify without sacrificing sensitivity, which eliminated the steps of nucleic acid extraction. Furthermore, by the CRISPR/Cas12a, the biosensor addressed false positives caused by non-specific amplification and had high sensitivity with the actual limit of detection (LOD) of 7.6×10-4 ng·µL-1 (4 copies·µL-1). In addition, the strategy was built on the lateral flow assay (LFA) strip to achieve visual and portable detection for point-of-care testing. Moreover, the biosensor by a fluorometer and LFA strip showed a high accuracy to rival qPCR in actual sample detection. Therefore, the biosensor is an ultra-sensitive and specific tool that can replace traditional methods. KEY POINTS: • No nucleic acid extraction, direct PCR-simplified steps, and reduced time and cost • CRISPR/Cas12a solved the false positives caused by nonspecific amplification • The combination of the LFA strip and biosensor is more convenient for POC detection.

Ultra-sensitive and point-of-care detection of Capripoxvirus (CaPV) based on loop-mediated amplification (LAMP) and trans-cleavage activity of CRISPR/Cpf1.

Capripoxvirus (CaPV) is one of the common skin diseases infecting cattle and sheep which can cause serious economic losses. Establishing ultra-sensitive, rapid, and point-of-care detection of CaPV is particularly important for hindering its spread. Here, we use the principle that CRISPR/Cpf1 can specifically recognize the target DNA and activate its trans-cleavage activity to identify the CaPV product amplified by loop-mediated amplification (LAMP). Under the designed specific primers, a set of LAMP which can amplify CaPV specifically was established and optimized firstly. Then, the CRISPR/Cpf1 was introduced to identify LAMP products. LAMP can be completed at a constant temperature, thus avoiding the use of temperature-variable instruments, making it possible to detect viruses outside the laboratory. To further satisfy the point-of-care detection of CaPV, we introduced a portable fluorometer and CRISPR-based lateral flow test. Due to the introduction of CRISPR/Cpf1, the sensitivity of the method is greatly increased, which is of great significance for the early detection of viruses. Through CRISPR/Cpf1-mediated fluorescence detection, we can detect CaPV as low as 1.47 × 10-3 TCID50 in 50 min, 1000 times more sensitive than quantitative real-time PCR. Through CRISPR-based lateral flow test, we can visually detect CaPV as low as 1.47 × 10-2 TCID50. Besides, this strategy can be used for the primary samples obtained from the cell culture of CaPV after simple ultrasonic disruption, which eliminates the complicated nucleic acid extraction steps required by traditional methods.

Analysis of vaccine-like lumpy skin disease virus from flies near the western border of China.

Lumpy skin disease (LSD) is a devastating viral disease that occurs in cattle. In China, it was first detected in the Xin-Jiang autonomous region, near the border with Kazakhstan, in August 2019. As there were no new occurrences of LSD in either country following the first detection, the initial introduction of the virus remains unknown. Arthropod vectors were considered as potential vectors. Consequently, to identify the arthropod vectors involved in transmitting LSD virus (LSDV), an insect surveillance campaign was launched at four different sites scattered along the border, and samples from 22 flying insect species were collected and subjected to PCR assays. Following the Agianniotaki LSDV vaccine and Sprygin's general LSDV assays, two kinds of non-biting flies, namely, Musca domestica L and Muscina stabulans, were positive for LSDV. However, all the other insects tested negative. Viral DNA was only detected in wash fluid, implying body surface contamination of the virus. The negative test results suggest that non-biting flies are the dominant insects involved in the observed local epidemic. Three genomic regions encoding RPO30, GPCR, and LW126 were successfully sequenced and subjected to phylogenetic analysis. The sequences shared high homology with LSDV/Russia/Saratov/2017, a recombinant vaccine-like strain formerly identified in Russia, and clustered with LSDV vaccine strains in phylogenetic trees of RPO30 and LW126. However, the GPCR gene was seen to be solely clustered with LSDV field strains, implying differences in host affinity between these closely related vaccine-like strains. Despite this, there is no direct evidence to support cross-border transmission of the vaccine-like LSDV. To our knowledge, this is the first report of vaccine-like LSDV DNA detection in non-biting flies in China.

Single-nucleotide variant of PIK3CA H1047R gene assay by CRISPR/Cas12a combined with rolling circle amplification.

PIK3CA H1047R gene plays an important role in the PI3K/Akt/mTOR signaling pathway, and its mutation is closely related to the occurrence and development of breast cancer and Lipoblastoma. Therefore, it is of great value to detect the PIK3CA H1047R mutant gene. Here, an analytical method coupled CRISPR/Cas12a with rolling circle amplification (RCA) technology was constructed for ultra-sensitive and specific detection of the single-nucleotide variant (SNV) of the PIK3CA H1047R gene. With efficient amplification of RCA and CRISPR/Cas12a, the detection limit of the mutant target and mixture of the mutant with wild-type target were as low as 10 aM and 0.036%, respectively. The detection limit of the RCA-CRISPR/Cas12a method was lower than that of allelic specific PCR (AS-PCR) for detecting SNV of the PIK3CA H1047R gene. Hence, this RCA-CRISPR/Cas12a method is sensitive and specific for the detection of SNV. What's more, this strategy provides a new idea for medical diagnosis and lays a technical foundation for the research of PI3K/Akt/mTOR signaling pathways.

Development of multiplex oligonucleotide microarray for simultaneous detection of six swine pathogens.

In order to establish a high-throughput identification technique that simultaneously detects six major pathogens including APP, HPS, PRRSV, Mhp, PCV-2 and CSFV, six pairs of primers and probes were designed based on the specific conservative sequences of the pathogens, a multiplex PCR system was developed, hybrid parameters were optimized, and evaluation of the technology was performed. The results showed that the present detection method had a sensitivity of 5.8 × 102copies/µL for APP, 7.8 × 103 copies/µL for HPS, 6.8 × 103 copies/µL for Mhp, 6.3 × 102 copies/µL for PCV-2, 4.8 × 103 copies/µL for PRRSV, and 5.5 × 102 copies/µL for CSFV, respectively; and it produced no cross reaction against the other nine pathogens like swine-origin pseudorabies virus, porcine parvovirus, Japanese B encephalitis virus, swine vesicular disease virus, vesicular stomatitis virus, foot-and-mouth disease virus, bluetongue virus, peste des petits ruminants virus and salmonella. Application of the multiplex oligonucleotide microarray established here to testing 285 clinical blood samples indicated a single infection rate of 18.2 % (52/285) and a mixed infection rate of 6.3 % (18/285) which were consistent with the results of the sequencing verification. This technique might serve as a rapid and high-throughput method of detection for epidemic investigation and clinical diagnosis of multiple pathogens.

Establishment of a Multiplex Real-Time TaqMan-MGB Polymerase Chain Reaction (PCR) Method for the Simultaneous Detection of Three Animal Chlamydia Species.

BACKGROUND Chlamydiae are spread globally and cause infectious diseases in both humans and animals. The existing detection methods for this disease have numerous shortcomings, including low sensitivity, time consuming procedures, and high contamination vulnerability. MATERIAL AND METHODS To overcome shortcomings for detecting animal chlamydiosis, a multiplex quantitative polymerase chain reaction (PCR) assay was established for simultaneously detecting and differentiating 3 Chlamydia species (C. pecorum, C. abortus, and C. psittaci) by real time PCR based on TaqMan-MGB technology. RESULTS The limit of detection was 20.2 copies/µL for Chlamydophila (Cp.) abortus, 30.8 copies/µL for Cp. pecorum, and 16 copies/µL for Cp. psittaci. This method has good repeatability and stability as coefficients of variation range from 0.04% to 1.38%. Furthermore, compared with OIE (World Organization for Animal Health) recommended PCR assay and previously reported animal chlamydia shell PCR, this multiplex PCR assay demonstrated 99% concordance in detecting clinical samples of porcine nasal swabs and vaginal swabs. CONCLUSIONS The novel established method in this study was able to detect 3 types of Chlamydia species simultaneously, and had high sensitivity, strong specificity, and good stability. It provided a rapid, reliable, and convenient method for epidemiological and clinical diagnosis of chlamydiosis in animals.

Combining reverse-transcription multiplex PCR and microfluidic electrophoresis to simultaneously detect seven mosquito-transmitted zoonotic encephalomyelitis viruses.

Several mosquito-transmitted viruses are causative agents for zoonotic encephalomyelitis. Rapid identification of these viruses in mosquito populations is an effective method for surveying these diseases. To detect multiple mosquito-transmitted viral agents, including West Nile virus, Saint Louis encephalitis virus, Venezuelan equine encephalomyelitis virus, Western equine encephalomyelitis virus, Eastern equine encephalomyelitis virus, Highlands J virus and Japanese encephalitis virus, an assay using multiplex reverse-transcription PCR combined with microfluidic electrophoresis was developed and evaluated. Tailed nested primers were used in the assay to amplify specific viral genomic segments, and products with specific length were further analyzed by using a microfluidic electrophoresis chip. The assay exhibited good specificity and analytical sensitivity (10(2) copies/µL). This technology can be helpful in the quarantine and surveillance of exotic encephalomyelitis viruses which are transmitted by mosquitoes.

Simultaneous typing of seven porcine pathogens by multiplex PCR with a GeXP analyser.

A novel high-throughput method was developed for simultaneous detection and differentiation of seven porcine pathogens by multiplex PCR based on a GenomeLab Gene Expression Profiler (GeXP) analyser. The pathogens included in this study were pseudorabies virus (PRV), classical swine fever virus (CSFV), African swine fever virus (ASFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine circovirus type 2 (PCV-2) and Japanese encephalitis virus (JEV). Seven pairs of chimeric primers, consisting of a pathogen-specific sequence fused to a universal sequence at the 5' end, were used to initiate the PCR, after which a set of universal primers was used for the subsequent cycles of the PCR. Amplification products were separated by capillary electrophoresis and identified using fluorescence spectrophotometry. The specificity of the GeXP assay was examined with single and mixed pathogen cDNA/DNA templates. The specific DNA product amplification peaks of seven pathogens were observed on the GeXP analyser. Negative controls did not produce DNA products. The sensitivity was evaluated by performing the assay on serial 10-fold dilutions of the plasmids containing the target sequence. Under optimised conditions this assay achieved a sensitivity of 100-1000 copies/µL for a single virus and 1000 copies/µL when all of the seven pre-mixed viral targets were present. Furthermore, the GeXP-PCR assay was 100% specific when 58 clinical samples were tested in comparison with the conventional PCR method. In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for simultaneously detecting seven pathogens that infect swine.

A nested multiplex polymerase chain reaction assay for the differential identification of three zooanthroponotic chlamydial strains in porcine swab samples.

Porcine chlamydial infection is an enzootic infectious disease caused by multiple members of the family Chlamydiaceae (e.g. Chlamydophila abortus, Chlamydia suis, and Chlamydophila pneumoniae). Rapid and accurate differentiation of these pathogens is critical in the control and prevention of disease. The aim of the current study was to develop a nested multiplex polymerase chain reaction (nmPCR) assay to simultaneously detect the 3 chlamydial pathogens in clinical samples. In the first round of the nmPCR, 1 pair of family-specific primers were used to amplify the 1,100 base pair (bp) fragment of chlamydial ompA gene. In the second round of the nmPCR, 4 inner primers were designed for Ch. abortus, C. suis, and Ch. pneumoniae. Each pathogen produced a specific amplicon with a size of 340 bp, 526 bp, and 267 bp respectively. The assay was sensitive and specific for detecting target pathogens in both cell cultures and clinical specimens. The results, incorporated with the improved rapid DNA extraction protocol, suggest that the nmPCR could be a promising assay for differential identification of different chlamydial strains in pigs.