A protocol for quantizing total bacterial 16S rDNA in plasma as a marker of microbial translocation in vivo

Increased systemic microbial translocation contributes to the pathogenesis of various diseases. The magnitude of microbial translocation is measured by total bacterial 16S ribosomal DNA (rDNA) in plasma using quantitative PCR (qPCR). An evaluation of human systemic microbial translocation in vivo is crucial for revealing microbial product-mediated inflammation, innate immune activation and immune perturbation. The human gut harbors 10¹² microorganisms per gram, and this is 10 times more than those from other sites.^1,2 The intestinal mucosal barrier prevents pathogen invasion and nonpathogenic antigens residing within the intestinal lumen.^1,2 Notably, the gut mucosal barrier prevents the host from being injured by pathogens, yet allows a very low level of bacterial product translocation to the system to maintain systemic immune homeostasis, as demonstrated by immune deficiencies in mice raised in sterile conditions.^3,4,5,6,7

A ‘leaky’ gut and increased systemic microbial translocation contribute to the pathogenesis of various diseases, such as HIV infection, type II diabetes and hepatitis.^8,9,10,11,12 We have previously shown that HIV infection causes increased systemic microbial translocation from a ‘leaky’ gut.^7,8,9 Increased levels of microbial translocation and its associated inflammation and immune activation play key roles in HIV disease progression, regardless of antiretroviral therapy.^7,8,9 Therefore, the quantification of total bacterial 16S rDNA in plasma is used to assess human and animal systemic microbial translocation in vivo, and thus is a great tool to study the role of systemic microbial products in disease pathogenesis and mucosal barrier function.

The bacterial 16S rDNA assay can analyze 90% of bacterial strains, including Gram-positive and Gram-negative bacteria.^8,13,14 However, use of this assay is highly challenging because of its high technical demands and the risk of contamination. Nonetheless, we have performed this assay successfully and have published several studies regarding the assay’s use.^3,7,8,15 A detailed protocol for quantifying total bacterial 16S rDNA in plasma is reported here.

Plasma sample preparation

Plasma samples were prepared as follows:

1. EDTA-containing tubes (catalog number: 366643, BD, Franklin Lakes, NJ, USA) were used to collect plasma samples.

2. Fresh blood samples and endotoxin-free water (a negative control) in EDTA-containing tubes were spun at 450g for 10 min, followed by transfer of the samples to new centrifuge tubes (catalog number: 352098, BD).

3. Samples were spun at 800g for 15 min.

4. Plasma and water were removed after two-step centrifugation, placed in aliquots and stored at −80 °C in a freezer. Low-binding centrifuge tubes (1.5 ml, catalog number: 80077-236, VWR, Radnor, PA, USA) were used to store plasma samples. Samples were thawed only once for the 16S rDNA assay because repeated freezing and thawing results in bacterial DNA degradation.

Isolation of microbiaL DNA from plasma samples

Microbial DNA was extracted from 400 μl of plasma and the water control using the QIAamp UCP pathogen Mini kit (catalog number: 50214, Qiagen, Valencia, CA, USA) according to the following steps:

1. A total volume of 40 μl of proteinase K was added to a 1.5 ml low-binding centrifuge tube (catalog number: 80077-236, VWR). Then, 400 μl of plasma (equilibrated to room temperature) or water was added. The lid was closed, and each sample was vortexed for 10 s.

2. Samples were incubated at 56 °C for 10 min.

3. A total volume of 200 μl of APL2 buffer was added. The lid was closed, and the samples were vortexed for 30 s.

4. Samples were incubated at 70 °C for 10 min.

5. The tubes were briefly spun.

6. A total volume of 300 μl of ethanol (catalog number: BP2818-500, Fisher Scientific, Pittsburgh, PA, USA) was added to each sample. The lid was closed, and the samples were vortexed for 15–30 s.

7. The 600 μl mixture from step 6 was carefully transferred into a QIAamp UCP Mini spin column in a 2 ml collection tube without wetting the rim. The lid was closed, and the mixture was spun at 6000g for 1 min. The column was transferred to a new 2 ml collection tube.

8. The remaining lysate from step 7 was added to the column. The lid was closed, and the mixture was spun at 6000g for 1 min. The column was transferred to a new 2 ml collection tube.

9. A total volume of 600 μl of APW1 buffer was carefully added without wetting the rim. The lid was closed, and the mixture was spun at 6000g for 1 min. The column was transferred to a new 2 ml collection tube.

10. A total volume of 750 μl of APW2 buffer was carefully added without wetting the rim. The lid was closed, and the mixture was spun at 20 000g for 3 min.

11. The column was transferred to a new 2 ml collection tube. The lid was closed, and the mixture was spun at 20 000g for 1 min.

12. The column was transferred to a new 2 ml collection tube. To dry the membrane completely, the lid was left open, and the tube was incubated at 56 °C for 3 min.

13. The column was transferred to a 1.5 ml low-binding centrifuge tube (catalog number: 80077-236, VWR). Then, 20 μl of AVE buffer was carefully added to the center of the QIAamp UCP membrane. The lid was closed, and the tube was incubated at room temperature for 1 min.

14. The tubes were spun at 20 000g for 1 min to elute the DNA.

15. The column was transferred to a 1.5 ml low-binding centrifuge tube. Then, 20 μl of AVE buffer (equilibrated to room temperature) was carefully added to the center of the QIAamp UCP membrane. The lid was closed, and the tubes were incubated at room temperature for 1 min.

16. The tubes were spun at 20 000g for 1 min to elute the DNA. The isolated DNA was qPCR-amplified immediately because freezing and thawing resulted in bacterial DNA fragment degradation.

qPCR for bacteriaL 16s rDNA

qPCR was performed according to the following steps:

1. Forward (8F: 5′--3′) and reverse (515R: 5′--3′) primers and probe (338P: 5′-FAM--BHQ1- 3′) were used for qPCR.

2. A 20 μl amplification reaction consisted of 10 μl of 2 × PerfeCTa qPCR ToughMix (catalog number: 95112, Quanta BioSciences, Gaithersburg, MD, USA), 0.3 μmol/l forward and reverse primers, 0.175 μmol/l probe and 5 μl of the extracted microbial DNA from plasma or the water control. Degenerate forward and reverse primers were used to amplify DNA templates encoding 16S rRNA.

3. Genomic DNA from bacterial E. coli (ATCC, Manassas, VA, USA) was used for generating standard curves. A standard curve was created by serial dilutions of PCR-amplified products containing known copy numbers of the template (Figure 1).

4. The DNA was amplified in duplicate to quadruplicate based on the assay performer’s technique accuracy, and mean or median values were calculated. The reaction conditions for DNA amplification were 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s and at 60 °C for 1 min.

Figure 1.

Standard qPCR curve for 16S rDNA amplification. qPCR, quantitative PCR; rDNA, ribosomal DNA.

Although gut histologic evaluation provides a direct assessment of permeability, the obvious advantage for clinical research in humans is to test systemic microbial translocation under in vivo conditions. Several methods are used to measure human systemic microbial translocation in vivo,^{8,10,16,17,18} including the assessment of gut permeability in vivo (the intestinal permeability (IP) test), plasma levels of lipopolysaccharide (LPS) and lipoteichoic acid (LTA), plasma levels of total bacterial 16S rDNA, and plasma levels of soluble CD14, intestinal fatty acid binding protein (I-FABP) and tight junctions (ZO-1, Claudin-2/4). The IP test directly assesses the intestinal permeability by analyzing the capacity of two nonmetabolized sugar molecules to permeate the intestinal mucosa.¹⁶ However, the IP test is affected by the sugar absorption, gut mucosal blood flow, kidney function and other factors related to gastrointestinal motility changes.^19,20,21 Plasma LPS of Gram-negative bacteria and LTA of Gram-positive bacteria cannot represent total bacterial products. All other methods are indirect assessments of bacterial products, and thus cannot accurately represent in vivo conditions. Therefore, it is advantageous to assess plasma total bacterial rDNA that is a direct measure of a majority of bacterial products in plasma. Thus, precise measurements of the plasma bacterial 16S rDNA are warranted.

Running all samples in one plate is an ideal way to compare cases and controls. However, when data are compared from more than one qPCR plate, removal of the between-plate variation is the key, even though the measurements were technically performed using identical procedures.²² The following key factors of the 16S rDNA assay are as follows. (1) A relatively sterile condition is always the key for controlling contamination; if the water control has more bacterial DNA than the human plasma samples, contamination can occur from either reagents, assay processing or the environment. (2) A low-DNA-binding tube is necessary to prevent target DNA binding to the tube wall. (3) Filtered low retention tips are necessary to prevent cross-contamination and to ensure the assay accuracy. (4) Two-step centrifugation is required for plasma isolation. (5) PerfeCTa qPCR ToughMix is required for qPCR amplification due to its high sensitivity for low amounts of the target template. (6) Isolated microbial DNA is required to conduct PCR amplification immediately without freezing and thawing.

Conflict of interest

The author declares no conflict of interest.