Kawashima Akira, Yanagawa Yasuaki, Chikata Takayuki, Shimogawara Rieko, Mizushima Daisuke, Tsuchiya Kiyoto, Yagita Kenji, Gatanaga Hiroyuki, Watanabe Koji
AIDS Clinical Center, National Center for Global Health and Medicine, Japan Institute for Health Security, Tokyo, Japan.
The Joint Research Center for Human Retrovirus Infection Kumamoto University Campus, Kumamoto, Japan.
PLoS Negl Trop Dis. 2025 Jun 5;19(6):e0012935. doi: 10.1371/journal.pntd.0012935. eCollection 2025 Jun.
TaqMan-probed quantitative PCR (qPCR) is highly valued for diagnosing Entamoeba histolytica infections (amebiasis). However, unclear cycle threshold (Ct) values often yield low-titer positive results, complicating interpretation. This study aimed to optimize qPCR primer-probe sets with logically determined cut-off Ct value using droplet digital PCR (ddPCR).
METHODOLOGY/PRINCIPAL FINDINGS: Amplification efficacy was evaluated using ddPCR by measuring absolute positive droplet counts (APD) and mean fluorescence intensity at different PCR cycles and annealing temperatures (AT). A primer-probe specific cut-off Ct value was determined from a standard curve by correlating Ct values with APD. Twenty primer-probe sets targeting small subunit rRNA gene regions (X64142) were designed from previous papers. Amplification efficacy remained consistent at high PCR cycles (50 cycles), but differed at lower PCR cycles (30 cycles), identifying five sets with higher amplification efficiency than other candidates. Of these, only two sets maintained efficiency at higher AT (62°C). Ct value was inversely proportional to the square of APD, defining the specific cut-off Ct value as 36 cycles. Selected primer-probe set with a cut-off effectively differentiated E. histolytica infection in clinical specimens. However, discordant results between Ct value and APD were seen in some cases with high Ct value. Shotgun metagenomic sequencing suggested microbial-independent false positive reactions contributed to these discrepancies, although specific reactants were unidentified.
CONCLUSIONS/SIGNIFICANCE: The combination use of ddPCR with qPCR revealed that false positive reactions of qPCR and/or ddPCR commonly happen in stool specimens. Also, this study emphasizes the value of ddPCR for establishing accurate cut-off values with efficient primer-probes.
TaqMan探针定量聚合酶链反应（qPCR）在诊断溶组织内阿米巴感染（阿米巴病）方面具有很高的价值。然而，不清楚的循环阈值（Ct）值常常产生低滴度阳性结果，使结果解读变得复杂。本研究旨在使用液滴数字PCR（ddPCR）优化qPCR引物-探针组，并合理确定截断Ct值。
方法/主要发现：通过在不同的PCR循环数和退火温度（AT）下测量绝对阳性液滴数（APD）和平均荧光强度，使用ddPCR评估扩增效率。通过将Ct值与APD相关联，从标准曲线确定引物-探针特异性截断Ct值。根据之前的文献设计了20组针对小亚基rRNA基因区域（X64142）的引物-探针。在高PCR循环数（50个循环）时扩增效率保持一致，但在较低的PCR循环数（30个循环）时有所不同，确定了五组扩增效率高于其他候选组的引物-探针。其中，只有两组在较高的AT（62°C）下仍保持效率。Ct值与APD的平方成反比，将特异性截断Ct值定义为36个循环。选定的带有截断值的引物-探针组有效地鉴别了临床标本中的溶组织内阿米巴感染。然而，在一些高Ct值的病例中，Ct值和APD之间出现了不一致的结果。鸟枪法宏基因组测序表明，尽管未鉴定出具体反应物，但微生物无关的假阳性反应导致了这些差异。
结论/意义：ddPCR与qPCR联合使用表明，qPCR和/或ddPCR的假阳性反应在粪便标本中普遍发生。此外，本研究强调了ddPCR在使用高效引物-探针建立准确截断值方面的价值。
