【摘要】：為研究海洋中常見(jiàn)致病性弧菌的快速分離富集方法——免疫磁珠法,本文以副溶血弧菌、哈維氏弧菌、創(chuàng)傷弧菌、燦爛弧菌為研究對(duì)象,制備納米免疫磁珠,并通過(guò)一系列優(yōu)化條件,用3M試紙計(jì)算捕獲率來(lái)確定免疫磁珠最佳捕獲條件。最后分別對(duì)4種致病性弧菌的不同菌液稀釋濃度、對(duì)1株目標(biāo)菌和8株非目標(biāo)菌、4種混合菌進(jìn)行捕獲,從而評(píng)價(jià)免疫磁珠的靈敏度、特異性以及抗干擾能力。結(jié)果表明,4種磁珠偶聯(lián)最佳抗體量為300、600、150、150μg。最佳捕獲條件:副溶血弧菌免疫磁珠用量3mg,免疫反應(yīng)45min,磁分離3min,p H 7.4緩沖液體系。哈維氏弧菌免疫磁珠用量3mg,免疫反應(yīng)45min,磁分離1min,p H 6.0緩沖液體系。創(chuàng)傷弧菌免疫磁珠用量12mg,免疫反應(yīng)60min,磁分離5min,p H 8.0緩沖液體系。燦爛弧菌免疫磁珠用量6mg,免疫反應(yīng)30min,磁分離3min,p H 7.4緩沖液體系。在純培養(yǎng)情況下,4種免疫磁珠靈敏度均達(dá)到10 CFU/m L數(shù)量級(jí);分別對(duì)9株菌進(jìn)行捕獲,對(duì)目標(biāo)菌的捕獲率均達(dá)到87%以上,對(duì)非目標(biāo)菌的捕獲率均低于32%;對(duì)4種混合菌進(jìn)行捕獲時(shí),免疫磁珠捕獲率不受其它雜菌干擾。本研究證明了該免疫磁珠有較好的靈敏度、特異性和抗干擾能力,且與其它增菌方法相比,大大縮短了時(shí)間,具有較好的應(yīng)用前景。
[Abstract]:In order to study the rapid isolation and enrichment of vibrio in the sea-immunomagnetic beads were prepared by using Vibrio parahaemolyticus, Vibrio Harvey's, Vibrio vulnificus and Vibrio splendens as research objects. Through a series of optimization conditions, the optimal capture conditions of immune beads were determined by calculating the capture rate of the 3M test paper. Finally, the dilution concentration of four pathogenic Vibrio vibrio strains in different liquid, one target bacterium, eight non-target bacteria and four mixed bacteria were captured to evaluate the sensitivity, specificity and anti-interference ability of the immune beads. The results showed that the best antibody concentration of four magnetic beads was 300600150150 渭 g. The optimal trapping conditions were as follows: the amount of immunomagnetic beads of Vibrio parahaemolyticus was 3 mg, the immune reaction was 45 min, and the magnetic separation was 3 min / h 7.4 buffer system. The dosage of immunomagnetic beads of Vibrio Harvey was 3 mg, the immune reaction was 45 min, and the magnetic separation was 1 min with pH 6.0 buffer system. The dosage of immunomagnetic beads of Vibrio vulnificus was 12 mg, the immune reaction was 60 min, and the magnetic separation was 5 min with pH 8.0 buffer system. The dosage of immunomagnetic beads of Vibrio splendens was 6 mg, the immune reaction was 30 min, and the magnetic separation was 3 mins / h 7.4 buffer system. In pure culture, the sensitivity of the four kinds of immunomagnetic beads was 10 CFU/m / L, 9 strains were captured, the capture rate of the target bacteria was more than 87%, and the capture rate of non-target bacteria was lower than 32%. The capture rate of immune beads was not disturbed by other hybrids when the four mixed bacteria were captured. This study proved that the immunomagnetic beads have better sensitivity, specificity and anti-interference ability, and compared with other methods of bacteriostasis, the time is greatly shortened, and has a better application prospects.
【作者單位】： 寧波大學(xué)海洋學(xué)院;
【基金】：海洋公益性行業(yè)科研專項(xiàng);201105007號(hào) 寧波大學(xué)研究生科研創(chuàng)新基金項(xiàng)目;G14045號(hào)
【分類號(hào)】：R155.5

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