Trojan Technologies, 3020 Gore Rd, London, ON, N5V 4T7, Canada.
Great Lakes Institute for Environmental Research, University of Windsor, 401 Sunset Avenue, Windsor, ON, N9B 3P4, Canada.
Water Res. 2019 Nov 1;164:114941. doi: 10.1016/j.watres.2019.114941. Epub 2019 Jul 31.
The most probable number dilution-culture assay (MPN) is used to enumerate viable phytoplankton in regulatory tests of ballast water treatment systems. However the United States Coast Guard has not yet accepted MPN, in part due to concerns of biased results due to cells being viable but not growing. MPN does not assess the fate of every cell, and thus the bias can only be evaluated by a companion method that assesses the ability of the various taxa to grow. This growth ability ("growability") is the complement of the bias, and has been evaluated by microscopic taxonomy of before-culture and after-culture samples. However, microscopic taxonomy is extremely laborious and few data have been produced for phytoplankton growability in MPN assays. To address the need for more and more reliable growability data, a method was developed using next-generation sequencing (NGS) and quantitative real time PCR (qRT-PCR) techniques that target the V9 region of the 18S rRNA gene for the taxonomic identification and growth assessment of eukaryotic phytoplankton, respectively. This growability method was applied to MPN samples from a ballast water management system test that were incubated with two different enrichment media at two different temperatures. DNA was extracted from filters of before-culture and after-culture samples, and assessed for taxonomy by NGS and for PCR template DNA concentration by qRT-PCR. Growth ratios based on changes in 18S template concentration over the incubation period were calculated for each taxon, and dead-cell DNA persistence through a 14 day incubation was verified to be <1% and did not influence the growth calculations. In total, 95 of 97 eukaryotic phytoplankton in the before-culture sample demonstrated growth, with definitive growth ratios ranging from 4.0 × 10-2.6 × 10. An additional 13 taxa demonstrated growth from non-detect in before-culture samples. Taxa-based growability values were 87-88% in individual incubation conditions with no statistical differences among conditions, and 98% for all conditions combined. When growability was weighted by the before-culture abundance of each taxa, relevant to regulations based on all organisms regardless of taxa, community-based growability was >99% in each condition and in all conditions combined because the most abundant taxa all exhibited growth. This study verifies that conventional phytoplankton MPN assays produce accurate results with low bias from undetected viable cells, regardless of enrichments and incubation temperatures. This work can provide regulatory confidence for broader acceptance of MPN assays without limitations.
最可能数稀释培养法（MPN）用于在压载水处理系统的监管测试中对浮游植物进行活细胞计数。然而，美国海岸警卫队尚未接受 MPN，部分原因是担心由于细胞存活但不生长而导致结果存在偏差。MPN 并未评估每个细胞的命运，因此只能通过评估各种分类群生长能力的辅助方法来评估这种偏差。这种生长能力（“可生长性”）是偏差的补数，并且已经通过培养前后样品的微观分类学进行了评估。然而，微观分类学非常繁琐，并且在 MPN 测定中浮游植物可生长性的数据很少。为了解决对更多更可靠可生长性数据的需求，开发了一种使用下一代测序（NGS）和定量实时 PCR（qRT-PCR）技术的方法，分别针对 18S rRNA 基因的 V9 区进行分类鉴定和真核浮游植物的生长评估。该可生长性方法应用于压载水管理系统测试的 MPN 样品，这些样品在两种不同的富集培养基中在两种不同的温度下孵育。从培养前和培养后样品的滤器中提取 DNA，并通过 NGS 进行分类鉴定，通过 qRT-PCR 进行 PCR 模板 DNA 浓度评估。根据孵育期间 18S 模板浓度的变化计算每个分类群的生长比，并验证在 14 天孵育过程中死亡细胞 DNA 的持久性<1%，并且不会影响生长计算。总共，在培养前样品的 97 种真核浮游植物中有 95 种显示出生长，确定的生长比范围为 4.0×10-2.6×10。另外 13 种在培养前样品中未检出的分类群显示出生长。在单独的孵育条件下，基于分类群的可生长性值为 87-88%，条件之间没有统计学差异，所有条件组合的可生长性值为 98%。当按每个分类群的培养前丰度对可生长性进行加权时，与基于所有生物而不考虑分类群的法规相关，在每种条件和所有条件组合中，群落可生长性均>99%，因为最丰富的分类群都表现出了生长。本研究证明，常规浮游植物 MPN 测定法即使在不进行富集且孵育温度不同的情况下，也能产生低偏差的准确结果，不存在未检测到的存活细胞。这项工作可以为更广泛地接受 MPN 测定法提供监管信心，而没有任何限制。
