黑斑壳病是一种尚未解决的疾病,会降低珍珠质量并威胁珍珠牡蛎的生存。在以往的研究中,细菌Tenacibaculumsp.从患病的Akoya珍珠牡蛎Pinctadafucata中分离出菌株Pbs-1,和一个快速的,具体,建立了灵敏的环介导等温扩增(LAMP)检测方法。该技术对于牡蛎孵化场和/或珍珠养殖场中的Pbs-1菌株的常规诊断具有相当大的潜力;因此,在环境样品中识别可能抑制LAMP的物质并找到可以减少LAMP抑制的添加剂是至关重要的。在这项研究中,我们调查了六种化学物质或蛋白质的影响,也称为常规PCR抑制剂,在灯上,使用菌株Pbs-1的DNA作为模板:腐殖酸,尿素,氯化铁(III)六水合物,黑色素,肌红蛋白,和乙二胺-N,N,N\',N'-四乙酸,二钠盐,二水合物(EDTA;pH6.5)。接下来,为了减少已确定的抑制剂的作用,我们测试了将牛血清白蛋白(BSA)或T4基因32蛋白(gp32)添加到LAMP测定中。当使用50ng的DNA模板时,4ng/μL腐殖酸,0.05%黑色素,10mM的EDTA(pH6.5)抑制LAMP反应,而肌红蛋白,尿素,和FeCl3没有影响。当使用50pg的DNA模板时,4ng/μL腐殖酸,0.05%黑色素,4μg/μL肌红蛋白,10μg/μL尿素,和10mMEDTA抑制LAMP反应。因此,研究表明,黑色素的基因扩增抑制作用,腐殖酸,可以通过向LAMP反应混合物中加入BSA或gp32来还原尿素。这项技术可以作为防止珍珠牡蛎大量死亡的协议的一部分;此外,结果增强了我们对抑制LAMP的物质和减少抑制的方法的了解,很少有报道。
Black-spot shell disease is an unresolved disease that decreases pearl quality and threatens pearl oyster survival. In previous studies, the bacterium Tenacibaculum sp. strain Pbs-1 was isolated from diseased Akoya pearl oysters Pinctada fucata, and a rapid, specific, and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting this pathogen was established. This technology has considerable potential for routine diagnosis of strain Pbs-1 in oyster hatcheries and/or pearl farms; therefore, it is vital to identify substances in environmental samples that might inhibit LAMP and to find additives that can reduce the inhibition. In this study, we investigated the effects of six chemicals or proteins, otherwise known as conventional PCR inhibitors, on LAMP, using the DNA of strain Pbs-1 as template: humic acid, urea, iron (III) chloride hexahydrate, melanin, myoglobin, and Ethylenediamine-N,N,N\',N\'-tetraacetic acid, disodium salt, dihydrate (EDTA; pH 6.5). Next, to reduce the effects of identified inhibitors, we tested the addition of bovine serum albumin (BSA) or T4 gene 32 protein (gp32) to the LAMP assay. When 50 ng of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, and 10 mM of EDTA (pH 6.5) inhibited the LAMP reaction, whereas myoglobin, urea, and FeCl3 had no effect. When 50 pg of DNA template was used, 4 ng/μL of humic acid, 0.05% melanin, 4 μg/μL of myoglobin, 10 μg/μL of urea, and 10 mM of EDTA inhibited the LAMP reaction. Thus, it was shown that the gene-amplification inhibitory effect of melanin, humic acid, and urea could be reduced by adding BSA or gp32 to the LAMP reaction mixture. This technique could be applied as part of a protocol to prevent mass mortalities of pearl oysters; moreover, the results enhance our knowledge about substances that inhibit LAMP and methods to reduce the inhibition, which have rarely been reported.