Detection of Frog Virus 3 by Integrating RPA-CRISPR/Cas12a-SPM with Deep Learning.

A fast, easy-to-implement, highly sensitive, and point-of-care (POC) detection system for frog virus 3 (FV3) is proposed. Combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a, a limit of detection (LoD) of 100 aM (60.2 copies/µL) is achieved by optimizing RPA primers and CRISPR RNAs (crRNAs). For POC detection, smartphone microscopy is implemented, and an LoD of 10 aM is achieved in 40 min. The proposed system detects four positive animal-derived samples with a quantitation cycle (Cq) value of quantitative PCR (qPCR) in the range of 13 to 32. In addition, deep learning models are deployed for binary classification (positive or negative samples) and multiclass classification (different concentrations of FV3 and negative samples), achieving 100 and 98.75% accuracy, respectively. Without temperature regulation and expensive equipment, the proposed RPA-CRISPR/Cas12a combined with smartphone readouts and artificial-intelligence-assisted classification showcases the great potential for FV3 detection, specifically POC detection of DNA virus.

Development of a triplex real-time PCR assay for detection and differentiation of gene-deleted and wild-type African swine fever virus.

African swine fever (ASF) is an infectious disease of domestic and wild pigs, caused by ASF virus (ASFV). In this study, a triplex real-time PCR assay was developed to detect and differentiate the gene-deleted and wild-type ASFV strains. Three pairs of primers and probes were designed to target the conserved region of B646L gene (p72), MGF_360-14L gene (located in the middle of MGF360-505R gene) and CD2v gene, respectively. Gene-deleted (with MGF360-505R and / or CD2v genes deletion) and wild-type ASFV strains were detected specifically and simultaneously by the assay developed without cross-reactions with other nucleic acids of PCV-2, CSFV, PRRSV, FMDV or SVA. The detection limits of the triplex rPCR were 7.9 copies, 9.7 copies, and 9.6 copies of standard plasmid DNA containing B646L gene, MGF_360-14L gene and CD2v gene, respectively. A total of 1215 field samples were tested in parallel by the triplex rPCR and real-time PCR recommended by OIE, and the B646L gene detection results were completely consistent between these two assays. The triplex rPCR assay was successfully developed to identify pigs infected with wild-type ASFV strains or immunized with the ASFV gene-deleted vaccine.

[Establish and optimization of real-time fluorescent reverse transcription loop-mediated isothermal amplification for detection of avian influenza H5 hemagglutinin gene].

H5 subtype avian influenza (AIV-H5) is a major causative agent of animalloimia a rapid and sensitive molecular biological diagnosis is crucial to the control program of AIV-H5. AIV-H5 real-time fluorescent reverse transcription loop-mediated isothermal amplification (qRT-LAMP) was established by means of heat treatment of the samples. The sensitivity, specificity and repeatability of this method were assessed and the performance of Calcein,SYBR Green I,HNB,SYTO 81 in colorimetric detection was comparatively analyzed to screen the optimum dye. The results showed the sensitivity of this method was 100 times higher than that of standard real-time fluorescent RT-PCR, and the detection limit was one copy of the gene per reaction. This method had no cross-reactivity with other common avian respiratory tract infectious disease-related pathogens such as IBV and NDV. The present study suggested Calcein was the optimum dye. Small-scale tests suggested this method was reliable for survey monitoring of AIV-H5 on the spot, indicating its potential applications in field investigation.

Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex).

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10⁻5(= 280ELD50) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10⁻4(=2800 ELD50). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.

Development and evaluation of an immunochromatographic strip for the detection of serum antibodies against bluetongue virus.

In this study, an immunochromatographic strip (ICS) was developed for the detection of bluetongue virus (BTV) serum antibodies. Colloidal gold particles labeled with streptococcal protein G (SPG), which can bind to the F(C) fragment of mammalian immunoglobulins, were used as the detector reagent. A recombinant VP7 BTV protein and a purified rabbit anti-SPG antibody were immobilized on test and control regions of a nitrocellulose membrane, respectively. In order to evaluate the ICS, 37 sera from animals exposed to different BTV serotypes were used as positive controls. In addition, 50 positive sera against viruses other than BTV, and eight sera taken from naive healthy sheep were used to determine the specificity of the ICS. Three hundred and three field sera taken from sheep and cattle were used after the above sera had been used for validation. Compared with the competitive ELISA (c-ELISA), the specificity and sensitivity of the ICS was 97.6% and 100%, respectively. There was excellent agreement between the results obtained by c-ELISA and the ICS (kappa=0.930). As it is rapid and easy to use, the test is suitable for the serological surveillance of BTV infection in the field.

[Multiplex fluorescent real-time PCR detection of bovine, goat and sheep derived materials in animal products].

We designed the specific primers and TaqMan probes targeting cytochrome b genes of mitochondrial DNA from bovine, goat and sheep. We used different fluorescents to label the probes. After optimization of reaction conditions, we set up a multiplex fluorescent real-time PCR method to detect bovine, goat and sheep derived materials, simultaneously. We finished the detection tests of 17 kinds of animal DNA and 200 DNA samples from different sources with the developed method and the National Standard GB/T 20190Y-2006 routine PCR method. The coincidence rate of these two methods was 100%. Without electrophoresis or restriction digestion, the developed method could reduce the test time to one third as routine PCR and identify three kinds of animal derived materials including bovine, goat and sheep in one reaction. The developed method was approximately 10 times more sensitive than routine PCR, and was applicable to identifications of bovine, goat and sheep derived materials in feed stuff, meat, milk, pelt and grease, etc. The study showed that the developed real-time PCR method is a rapid, sensitive and efficacious detection assay for bovine, goat and sheep derived materials in animal products.

[Influence of cry2A sporulation-dependent promoter and molecular chaperone ORF1-ORF2 from Bacillus thuringiensis on Cry11Aa protein].

OBJECTIVE: To analyze the coordination function of cry2A sporulation-dependent promoter and the enhanced expression of molecular chaperone ORF1-ORF2 to crystal protein Cry11Aa. METHODS: We constructed three recombinant plasmids pHcy1, pHcy2 and pHcy4 containing cry11Aa gene. pHcy1 carried cry11Aa own promoter and p19 gene, and pHcy2 carried cry2A sporulation-dependent promoter and orf1-orf2 gene. pHcy4 inserted cry2A promoter and orf1-orf2 gene upstream pHcy1 plasmid. The recombinant plasmids were introduced into an acrystalliferous mutant 4Q7 of Bacillus thuringiensis sub sp. israelensis. We performed SDS-PAGE to analyze Cry11Aa protein expression in the recombinant Bt strains and carried out the mosquitocidal bioassay. RESULTS: SDS-PAGE showed that Cry11Aa protein was detected in 4Q7(pHcy1) and 4Q7(pHcy4), but not in 4Q7(pHcy2). The cry11Aa gene could not be regulated under cry2A promoter. Cry11Aa protein had a 1.25 fold expression amount in the equal volume culture of 4Q7(pHcy4) to that of 4Q7(pHcy1), which indicated that molecular chaperone ORF1-ORF2 could enhance Cry11Aa expression amount to a certain extent. Both 4Q7(pHcy1) and 4Q7(pHcy4) formed Cry11Aa crystals in a similar size and shape during sporulation under the transmission electron microscope. Their LC50s against 3rd-instar Culex quinquefasciatus were 59.33 ng/mL and 66.21 ng/mL respectively. CONCLUSION: Whether crystal protein from B. thuringiensis could successfully express might relate to the type of the used promoter and their coordination. Molecular chaperone ORF1-ORF2 could enhance Cry11Aa expression amount to a certain extent with an unknown mechanism, but did not have an effect on high mosquitocidal toxicity of Cry11Aa protein. This research might play an important role to search the best collocation between ICP promoter or chaperone gene and ICP gene and to construct high-toxic Bacillus thuringiensis engineering strain by chaperone gene.

[Influence of accessory protein P19 from Bacillus thuringiensis on insecticidal crystal protein Cry11Aa].

p19 gene, cry11Aa gene and p20 gene from Bacillus thuringienesis subsp. israelensis are organized as a single operon. It is reported that P20 polypeptide is not required for high-level expression of Cry11Aa and crystal formation in B. thuringiensis. It is deduced that P19 might relate to Cry11Aa crystallization. In this study, two recombinant plasmids pHcy1 and pHcy3 containing cryllAa gene were constructed, the latter absent from p19 gene encoding a possible accessory protein between cry11Aa promoter and cry11Aa gene. The recombinant plasmids were introduced into an acrystalliferous mutant 4Q7 of B. thuringiensis subsp. israelensis. SDS-PAGE showed that Cry11Aa protein per unit of culture medium had a higher expression level in 4Q7(pHcy1) with p19 and cry11Aa genes than in 4Q7(pHcy3) with only cry11Aa gene. Both two B. thuringiensis strains formed Cry11Aa crystals in a similar size and shape during sporulation. Toxicity bioassay showed 4Q7 (pHcy1) and 4Q7 (pHcy3) exhibited a comparable mosquito-larvicidal activity against 3rd-instar Culex quinquefasciatus. It indicated that accessory protein P19 did not have an effect on cry11Aa crystallization and high mosquitocidal toxicity. However, it could enhance Cry11Aa expression amount to a certain extent.

[Bacillus thuringiensis helper protein P20 affects the formation of Cry1Ab].

The Cry1Ab differs most significantly from the other related ICPs by its absence of a carboxyl terminus of 28 amino acids including four cysteines; consequently it is less stable. We report that the helper protein P20 plays a role in the expression and crystallization of Cry1Ab. Three Cry1Ab expression plasmids pT1B, pP1B, and pDP1B, were constructed based on the shuttle vector pHT3101. The vector pT1B does not contain the p20 gene, pP1B carries p20, and pDP1B contains p20 with cry1A(c) promoter. Transformants were obtained by electroporating the plasmids into Bacillus thuringiensis acrystalliferous mutant CryB. Western blot demonstrated that crylAb was expressed as a 130 kD protein in all the transformants, and some of the protein was partially degraded into a 60 kD peptide. Quantitative protein analysis indicated that the amount of the 130 kD protein varied in the transformants and was in the ratio of 1:1.4:1.5 for PT1B, pP1B and pDP1B respectively. For the 60 kD proteins, the ratio was 1:1.1:1.6. Microscopic examination revealed that the size of the typical pyramidal crystals in the three transformants was in the order of T1B < P1B < DP1B. Bioassay showed that T1B, P1B and DP1B were all toxic to the larvae of Helicoverpa armigera with similar LC50. This study suggested that P20 plays a role in the expression and crystallization of Cry1Ab.

Cloning and expression of cyt2Ba7 gene from a soil-isolated Bacillus thuringiensis.

Bacillus thuringiensis ( Bt) cyt genes coding hemolytic and cytolytic toxins constitute a gene family, which are divided into two groups: cyt1 and cyt2. A novel cyt2 gene was detected from a soil-isolated Bt strain T301, which was highly homologous to cyt2Ba1 and finally designated cyt2Ba7. Until now, Cyt2Ba has not been expressed alone in Bt or other hosts. In this study, the cyt2Ba7 gene was cloned into the vector pQE30 and expressed as a fusion protein with 6xHistidine residues in Escherichia coli. Unlike cyt1A, cyt2Ba7 was freely expressed and formed cytoplasmic inclusions without the need for a "helper" protein. The 6xHis-tagged Cyt2Ba7 was purified in one step by Ni-NTA affinity chromatography, examined cytolytic activity on Sf9 cells, and developed as an antigen to obtain the antiserum against Cyt2Ba by subcutaneous injection into rabbits. This gene was also cloned into the Bt-E. coli shuttle vector pHT3101 and expressed in Bt strain 4Q7. Immunoblotting analysis revealed that the antiserum was remarkably selective and specific to Cyt2Ba.

Expression of the full-length and 3'-spliced cry1Ab gene in the 135-kDa crystal protein minus derivative of Bacillus thuringiensis subsp. kyushuensis.

Bacillus thuringiensis produces a 130-135-kDa insecticidal protein in the form of bipyramidal crystal which is toxic to lepidopteran larvae. Part of the C-terminal region of the native Cry1Ab was replaced by a heterologous sequence of Cry11Aa C-terminus to get a 3'-spliced cry1Ab gene. The full-length cry1Ab and 3'-spliced cry1Ab, which were both cloned into the E. coli-B. thuringiensis shuttle expression vector pHZB1, were expressed in a 135-kDa crystal protein minus derivative of B. thuringiensis subsp. kyushuensis (4U1-Cry(-135)). The crystal shape of Cry1Ab proteins from both recombinants was regularly bipyramidal, while the crystal size of the intact Cry1Ab was approximately fivefold larger than the 3'-spliced Cry1Ab. In addition, these two kinds of Cry1Ab proteins had similar toxicity against Argyrogramma agnata larvae.