[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.

[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.