液滴數(shù)字聚合酶鏈?zhǔn)椒磻?yīng)芯片及其在致病菌檢測(cè)中的應(yīng)用
發(fā)布時(shí)間：2018-09-05 14:04
【摘要】：設(shè)計(jì)與制作了一種基于聚二甲基硅氧烷-玻璃(PDMS-Glass)的多功能集成式液滴數(shù)字聚合酶鏈?zhǔn)椒磻?yīng)(ddPCR)芯片,該芯片由產(chǎn)生液滴的PDMS模塊和收集液滴的玻璃腔體模塊組成。PDMS模塊采用雙通道的T形結(jié)構(gòu)設(shè)計(jì),液滴產(chǎn)生速度快且通量高,在30 min內(nèi)可生成2×10~6個(gè)直徑約為20μm的微液滴。玻璃腔體模塊中存儲(chǔ)的液滴在整個(gè)實(shí)驗(yàn)過程中無需轉(zhuǎn)移,可直接在原位PCR儀上進(jìn)行擴(kuò)增,每個(gè)液滴均是一個(gè)微反應(yīng)器,經(jīng)過多次熱循環(huán)后,液滴仍能保持良好的穩(wěn)定性。選用副溶血性弧菌(VP)作為食源性致病菌的研究模型,考察了ddPCR芯片對(duì)其基因組DNA的絕對(duì)定量能力,結(jié)果表明,該ddPCR芯片對(duì)VP基因組DNA絕對(duì)定量的線性范圍寬,可跨越5個(gè)數(shù)量級(jí)(10~1~10~6 copies/μL),定量結(jié)果與DNA理論參考濃度間有很好的相關(guān)性。
[Abstract]:A multifunctional integrated liquid drop digital polymerase chain reaction (ddPCR) chip based on polydimethylsiloxane glass (PDMS-Glass) was designed and fabricated. The chip is composed of droplet generation PDMS module and glass cavity module. PDMS module is designed with a two-channel T-shaped structure. The droplet can produce 2 脳 10 ~ 6 microdroplets with a diameter of about 20 渭 m in 30 min. The droplets stored in the glass chamber module can be amplified directly on the in-situ PCR instrument without transfer during the whole experiment. Each droplet is a microreactor. After several thermal cycles, the droplet can still maintain good stability. Vibrio parahaemolyticus (VP) was selected as the research model of foodborne pathogenic bacteria. The absolute quantitative ability of ddPCR chip to its genomic DNA was investigated. The results showed that the linear range of absolute quantification of ddPCR chip to VP genome DNA was wide. There is a good correlation between the quantitative results of 5 orders of magnitude (10 ~ (-1) ~ 10 ~ (6) copies/ 渭 L), and the theoretical reference concentration of DNA.
【作者單位】： 上海海洋大學(xué)食品學(xué)院;傳感技術(shù)國家重點(diǎn)實(shí)驗(yàn)室中國科學(xué)院上海微系統(tǒng)與信息技術(shù)研究所;農(nóng)業(yè)部水產(chǎn)品貯藏保鮮質(zhì)量安全風(fēng)險(xiǎn)評(píng)估實(shí)驗(yàn)室(上海);上海海洋大學(xué)食品學(xué)院食品熱加工工程技術(shù)研究中心;
【基金】：國家自然科學(xué)基金(21405167,81472751,61271162) 上海晨光計(jì)劃(15CG54) 國家科技重大專項(xiàng)(2017ZX10302201-005-006)
【分類號(hào)】：R155.3
，
本文編號(hào)：2224498
[Abstract]:A multifunctional integrated liquid drop digital polymerase chain reaction (ddPCR) chip based on polydimethylsiloxane glass (PDMS-Glass) was designed and fabricated. The chip is composed of droplet generation PDMS module and glass cavity module. PDMS module is designed with a two-channel T-shaped structure. The droplet can produce 2 脳 10 ~ 6 microdroplets with a diameter of about 20 渭 m in 30 min. The droplets stored in the glass chamber module can be amplified directly on the in-situ PCR instrument without transfer during the whole experiment. Each droplet is a microreactor. After several thermal cycles, the droplet can still maintain good stability. Vibrio parahaemolyticus (VP) was selected as the research model of foodborne pathogenic bacteria. The absolute quantitative ability of ddPCR chip to its genomic DNA was investigated. The results showed that the linear range of absolute quantification of ddPCR chip to VP genome DNA was wide. There is a good correlation between the quantitative results of 5 orders of magnitude (10 ~ (-1) ~ 10 ~ (6) copies/ 渭 L), and the theoretical reference concentration of DNA.
【作者單位】： 上海海洋大學(xué)食品學(xué)院;傳感技術(shù)國家重點(diǎn)實(shí)驗(yàn)室中國科學(xué)院上海微系統(tǒng)與信息技術(shù)研究所;農(nóng)業(yè)部水產(chǎn)品貯藏保鮮質(zhì)量安全風(fēng)險(xiǎn)評(píng)估實(shí)驗(yàn)室(上海);上海海洋大學(xué)食品學(xué)院食品熱加工工程技術(shù)研究中心;
【基金】：國家自然科學(xué)基金(21405167,81472751,61271162) 上海晨光計(jì)劃(15CG54) 國家科技重大專項(xiàng)(2017ZX10302201-005-006)
【分類號(hào)】：R155.3
，
本文編號(hào)：2224498
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