Buongiorno Joy, Turner Stephanie, Webster Gordon, Asai Masanori, Shumaker Alexander K, Roy Taylor, Weightman Andrew, Schippers Axel, Lloyd Karen G
Department of Microbiology, University of Tennessee, Knoxville, TN 37996, USA.
Geomicrobiology, Federal Institute for Geosciences and Natural Resources (BGR), Hannover 30655, Germany.
FEMS Microbiol Ecol. 2017 Mar 1;93(3). doi: 10.1093/femsec/fix007.
Two common quantification methods for subseafloor microorganisms are catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) and quantitative PCR (qPCR). Using these methods, we quantified Bacteria and Archaea in Baltic Sea basin sediments (IODP Exp. 347) down to 90 mbsf, testing the following hypotheses in an interlaboratory comparison: (1) proteinase K permeabilization of archaeal cell walls increases CARD-FISH accuracy and (2) qPCR varies by more than an order of magnitude between laboratories using similar protocols. CARD-FISH counts did not differ between permeabilization treatments, demonstrating that proteinase K did not increase accuracy of CARD-FISH counts. However, 91% of these counts were below the quantification limit of 1.3 × 107 cells cm-3. For qPCR, data varied between laboratories, but were largely within the same order of magnitude if the same primers were used, with 88% of samples being above the quantification limit. Copy number values were elevated by preparing a sediment slurry before DNA extraction: 3.88 × 106-2.34 × 109 16S rRNA gene copies cm-3 vs. 1.39 × 107-1.87 × 109 total cells cm-3. By qPCR, Bacteria were more abundant than Archaea, although they usually were within the same order of magnitude. Overall, qPCR is more sensitive than CARD-FISH, but both require optimization to consistently achieve both precision and accuracy.
海底微生物的两种常见定量方法是催化报告沉积荧光原位杂交（CARD-FISH）和定量PCR（qPCR）。我们使用这些方法对波罗的海盆地沉积物（综合大洋钻探计划第347航次）中至90米海底以下深度的细菌和古菌进行了定量，在一项实验室间比较中检验了以下假设：（1）蛋白酶K对古菌细胞壁的通透作用可提高CARD-FISH的准确性；（2）使用相似方案的不同实验室之间，qPCR结果的差异超过一个数量级。通透处理之间的CARD-FISH计数没有差异，表明蛋白酶K并未提高CARD-FISH计数的准确性。然而，这些计数中有91%低于1.3×10⁷个细胞/立方厘米的定量限。对于qPCR，不同实验室的数据有所不同，但如果使用相同引物，数据大多在同一数量级内，88%的样本高于定量限。通过在DNA提取前制备沉积物浆液，拷贝数有所提高：16S rRNA基因拷贝数为3.88×10⁶ - 2.34×10⁹个/立方厘米，而总细胞数为1.39×10⁷ - 1.87×10⁹个/立方厘米。通过qPCR检测，细菌比古菌更为丰富，尽管它们通常处于同一数量级。总体而言，qPCR比CARD-FISH更灵敏，但两者都需要优化以始终如一地实现精确性和准确性。
