Abstract：
In the Japanese official detection method for unauthorized genetically modified (GM) papayas, one of two types of real-time PCR reagents with DNA polymerase (TaqMan Gene Master Mix [TaqMan Gene] or FastGene QPCR Probe Mastermix w/ROX [FastGene]) is primarily used for measurement. In 2022, we conducted a laboratory performance study on the unauthorized GM papaya line PRSV-YK, and the results revealed that high threshold cycle (Cq) values for the PRSV-YK detection test were obtained using TaqMan Gene with the 7500 Fast & 7500 Real-Time PCR System (ABI7500) and QuantStudio 12K Flex (QS12K), indicating the possibility of false negatives. The possibility of similar problems with all unauthorized GM papaya lines detection tests needs to be evaluated. In this study, we performed detection tests on unauthorized GM papaya lines (PRSV-YK, PRSV-SC, and PRSV-HN), the cauliflower mosaic virus 35S promotor (CaM), and a papaya positive control (Chy), and examined how the limits of detection (LOD) for each test are affected by two types of DNA polymerases (TaqMan Gene and FastGene) and three types of real-time PCR instruments (ABI7500, QS12K, and LightCycler 480 Instrument II [LC480]). In the PRSV-YK and PRSV-SC detection tests using ABI7500 and QS12K, measurement with TaqMan Gene showed a higher LOD than FastGene. In this case, an exponential amplification curve was confirmed on the amplification plot; however, the amplification curve did not cross the ΔRn threshold line and the correct Cq value was not obtained with a threshold line=0.2. The other tests (PRSV-HN, CaM, and Chy with ABI7500 and QS12K, and all detection tests with LC480) showed no important differences in the LOD for each test using either DNA polymerase. Therefore, when performing PRSV-YK and PRSV-SC detection tests with the ABI7500 or QS12K, FastGene should be used to avoid false negatives for foods containing GM papaya lines PRSV-YK and PRSV-SC at low mixing levels.
摘要：
在日本官方检测未经授权的转基因（GM）木瓜的方法中，具有DNA聚合酶的两种类型的实时PCR试剂之一（TaqMan基因主混合物[TaqMan基因]或FastGeneQPCR探针Mastermixw/ROX[FastGene]）主要用于测量。2022年，我们对未经授权的转基因木瓜品系PRSV-YK进行了实验室性能研究，结果表明，使用TaqMan基因与7500Fast和7500Real-TimePCR系统（ABI7500）和QuantStudio12KFlex（QS12K）获得了PRSV-YK检测测试的高阈值循环（Cq）值，表明假阴性的可能性。需要评估所有未经授权的GM木瓜线检测测试出现类似问题的可能性。在这项研究中,我们对未经授权的转基因木瓜品系(PRSV-YK，PRSV-SC,和PRSV-HN），花椰菜花叶病毒35S启动子（CaM），和木瓜阳性对照（Chy），并检查了每种测试的检测限（LOD）如何受到两种类型的DNA聚合酶（TaqMan基因和FastGene）和三种类型的实时PCR仪器（ABI7500，QS12K，和LightCycler480仪器II[LC480])。在使用ABI7500和QS12K的PRSV-YK和PRSV-SC检测试验中，用TaqMan基因测量显示比FastGene更高的LOD。在这种情况下,在扩增图上证实了指数扩增曲线；然而，扩增曲线没有越过ΔRn阈值线,并且在阈值线=0.2的情况下没有获得正确的Cq值。其他测试（PRSV-HN，CaM,和Chy与ABI7500和QS12K，使用LC480）进行的所有检测测试均显示，使用两种DNA聚合酶进行的每次测试的LOD均无重要差异。因此,用ABI7500或QS12K进行PRSV-YK和PRSV-SC检测测试时，FastGene应用于避免在低混合水平下含有GM木瓜系PRSV-YK和PRSV-SC的食物的假阴性。
