Institution of a digital PCR technique for detection of Borrelia burgdorferi sensu lato complicated DNA in cerebrospinal fluid


dPCR optimization workflow

An in depth description of the experimental setups is introduced in Supplementary. Our method to ascertain and optimise a extremely particular and delicate Borrelia dPCR technique are outlined in Fig. 1 and described under.

Determine 1
figure 1

Workflow of the stepwise experimental setups throughout institution of the Borrelia-specific digital PCR (dPCR) assay. The workflow highlights the completely different phases that have to be carried out to judge the robustness, specificity, and sensitivity of the Borrelia dPCR assay and pre-PCR technique. The Roman numerals point out the order of the experiments. (A) Institution and validation of the in-house Borrelia dPCR assay. (B) Optimization of “direct” pre-PCR technique with out DNA purification. (C) Comparability of “direct” technique to Maxwell DNA purification technique. (D) Take a look at of affected person samples with optimized pre-analytical technique and dPCR assay.

Borrelia dPCR assay institution and optimization (A)

For detection of the 5 Borrelia species (B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. spielmanii, and B. bavariensis) identified to trigger LNB, we selected to make use of the Borrelia-specific primer and probe sequences described by Ornstein and Barbour in 2006. We carried out an in-silico verification of the primers and probe in two phases. First, primers and probes had been entered into the NCBI Nucleotide BLAST internet utility and checked in opposition to the nucleotide assortment database (nr/nt) to search for sequence homology with completely different Borrelia strains in addition to potential non-specific matching of significantly human or microbial origin. BLAST default settings had been utilized. Second, the primers and probes had been checked for oligo-dimerization utilizing the IDT OligoAnalyzer™ Instrument (Built-in DNA Applied sciences Inc., USA). All primers and probes fulfilled our acceptance standards, e.g., “Count on worth”21 for sequence homology under 0.01.

In the course of the preliminary dPCR experiments we noticed that it was vital to incubate the dPCR plates on the PCR thermal cycler at 4 °C, in a single day, previous to studying to acquire a minimal of 15,000 droplets per nicely. When evaluating the full droplet counts from plates that had been learn immediately after PCR amplification (imply 13,519 droplets per nicely) to the full droplet counts from incubated plates (imply 17,524 droplets per nicely), the distinction was statistically important (p < 0.001).

The singleplex Borrelia dPCR assay was optimised utilizing a stepwise method to judge how the completely different parameters (Fig. 1A-III to A-V) affected the clustering (grouping of both optimistic or adverse partitions), cluster separation (differentiation between optimistic and adverse partitions), the quantity of rain, and the quantification. Consultant knowledge from these experiments will be seen in Fig. 2a–c.

Determine 2
figure 2

Examples of digital PCR (dPCR) output demonstrating the optimization of the Borrelia burgdorferi sensu lato dPCR assay. The identical assay elements and Borrelia gBlock template is utilized in all elements of the determine until in any other case acknowledged. Every column represents samples from particular person wells and is separated by a gray line. Blue dots characterize the optimistic partitions and gray dots characterize the adverse partitions. The horizontal pink traces illustrate the guide threshold setting for the info evaluation. Amplitude on Y-axis refers to fluorescence depth of particular person partitions. NTC (non-template management) was molecular graded water in all experiments. All figures present one representing PCR response of every pattern within the experiment until acknowledged in any other case. (a) The impact of the annealing temperature (gradient from 54 to 64 °C) on the amplification, and thus on the ultimate amplitude of the optimistic and adverse partitions. For every examined temperature, PCR samples containing NTC or roughly 600 copies of Borrelia gBlock had been run in duplicates. (b) The impact of various concentrations of ahead and reverse primers, on the distinction in fluorescence amplitude between optimistic and adverse partitions. The probe focus was 200 nM in each PCR response. For every mixture examined, PCR samples containing NTC or roughly 600 copies of Borrelia gBlock had been run in triplicates. (c) The impact of various probe concentrations on the fluorescence depth. PCR reactions contained 900 nM ahead and 900 nM reverse primer. For every mixture examined, PCR samples containing NTC or roughly 600 copies of Borrelia gBlock had been run in triplicates. (d) Twofold dilution sequence for evaluation of the analytical sensitivity. PCR reactions contained 900 nM ahead and reverse primer and 300 nM probe. For every dilution step examined, PCR samples containing NTC or Borrelia gBlock had been measured in eightfold.

There was no distinction when evaluating temperatures between 54 and 62 °C (Fig. 2a). Nevertheless, at 64 °C, the fluorescent amplitude distinction between optimistic and adverse partitions was markedly decreased. The optimum annealing temperature for the PCR amplification was decided to be 62 °C, to realize the best diploma of goal specificity as doable together with optimum cluster separation.

To find out the optimum concentrations of the ahead and reverse primers, we examined completely different primer pair combos of 4 ahead primer concentrations (300–1200 nM) with 4 reverse primer concentrations (300–1200 nM) in a primer matrix. The ensuing knowledge (Fig. 2b) was extracted from the QuantaSoft software program to Excel. The imply amplitude of the adverse clusters was subtracted from the imply amplitude of the optimistic clusters to calculate the imply amplitude distinction. The primer pair with the best imply amplitude distinction was 900 nM ahead primer together with 900 nM reverse primer (Supplementary A, Desk SA6).

The optimum probe focus was decided utilizing combos of probe concentrations starting from 50 to 600 nM, all examined in triplicates. Primarily based on visible inspection of the outcomes, 300 nM probe was decided to greatest separate the optimistic and adverse partitions (Fig. 2c).

A fraction of the Phocid herpesvirus (PhHV) was used as goal for the interior optimistic PCR management (IPC). First, the IPC assay was examined alone to evaluate the separation of the IPC optimistic cluster from the IPC adverse cluster (knowledge not proven). Then the Borrelia and the IPC assays had been mixed in a duplex to judge whether or not the assays affected one another. The analysis was carried out utilizing an IPC focus of 500 IPC copies per PCR response and excessive (500 copies), medium (50 copies) and low (5 copies) of Borrelia gBlocks per PCR response (Supplementary A Fig. SA1). We didn’t observe any distinction relating to amplitude separation, rain or quantification of Borrelia gBlock targets, when evaluating the Borrelia/IPC duplex assay to the Borrelia singleplex assay.

False-positive partitions are identified to happen in dPCR experiments, as a consequence of underlying Taq errors. Subsequently, we assessed the frequency of false-positive partitions by testing samples containing solely PCR-grade water, the “adverse CSF matrix”, or Maxwell elution buffer (Supplementary A, Fig. SA2). No false-positive partitions had been detected within the samples with “adverse CSF matrix” or Maxwell elution buffer, nevertheless in 5% (1/20) of the adverse samples with PCR-grade water we noticed one false-positive partition. Subsequently, we decided that for all subsequent evaluation, samples had been to be run as duplicates and {that a} pattern may solely be labeled as optimistic if (I) one of many duplicates had not less than two optimistic partitions, or (II) if each duplicates had not less than one optimistic partition every with the identical fluorescence amplitude because the optimistic cluster of the optimistic management within the Borrelia assay.

The analytical reactivity describes the efficiency of the dPCR assay by way of cross-reactivity and skill to detect the specified targets (specificity), along with an evaluation of restrict of detection (sensitivity). The analytical specificity was evaluated utilizing a group of purified bacterial and viral DNA from both laboratory tradition strains or QCMD previous panels, representing varied vital differential diagnostic CNS infections (Supplementary A, Desk SA4) and a group of Borrelia reference strains (Supplementary A, Desk SA5). We didn’t detect any false-positives samples and the Borrelia-specific dPCR assay did detect all Bb reference strains (knowledge not proven). The analytical sensitivity of the Borrelia assay was decided by a twofold dilution sequence utilizing identified copies of Borrelia gBlock in water (Fig. 2D). The restrict of detection (LOD) was outlined because the lowest variety of Borrelia copies per PCR response, the place not less than two partitions had been current in minimal 4 of the eight PCR replicates, or equal in not less than eight optimistic partitions within the pooled eight replicates. The estimated analytical sensitivity was 4 copies of Borrelia gBlock within the PCR response in not less than 15,000 whole partitions. Decrease quantities of Borrelia copies per PCR response had been additionally detectable, however at a extra rare price.

Optimization of “direct” pre-PCR technique with out DNA extraction (B)

dPCR has been described to be much less liable to PCR interference by matrix substances. Subsequently, we optimized a way for direct detection of Bb spiked in adverse CSF matrix. First, the spiked samples had been boiled at 95 °C, cooled to 4 °C, after which the crude extracts had been assessed by Borrelia dPCR as described intimately in Supplementary B. The assay was capable of detect the launched Borrelia DNA goal (Fig. 3a) in concentrations all the way down to 100 Bb per pattern. A centrifugation step previous to boiling resulted in improved detection of Bb per pattern (Fig. 3b) all the way down to 10 Bb per pattern, but it surely additionally induced extra intermediate fluorescent partitions (termed rain). Each the improved sensitivity and the emerged rain had been outcomes of ten-fold focus of the pattern by the centrifugation step. To scale back the quantity of rain occurring after the focus step, we evaluated two approaches to reduce PCR inhibition, a quick-spin step, and a proteinase Ok remedy respectively. The short-spin step successfully decreased the quantity of rain and facilitated a great separation of the optimistic and adverse clusters (Fig. 3c). The proteinase Ok remedy didn’t take away a lot of the intermediate partitions (Fig. 3d). Subsequently, the quick-spin step was chosen as most popular technique for rain discount. Thus, the optimized direct technique for detection of remoted Borrelia DNA with out DNA extraction was as follows; centrifugation adopted by boiling and cooling, and lastly a quick-spin previous to dPCR mixing and evaluation.

Determine 3
figure 3

Optimization of a “direct” pre-PCR technique for detection of Borrelia DNA in CSF matrix with out purification introduced in a 2D-plot. The blue and inexperienced dots characterize the Borrelia optimistic partitions and IPC optimistic partitions respectively, and gray dots characterize the adverse partitions. The horizontal pink traces illustrate the guide threshold setting for the info evaluation. Amplitude on X and Y-axis refers to fluorescence depth of particular person partitions. For each spike experiment Borrelia garinii pressure Lu118 was counted and diluted with “adverse CSF matrix”. Every determine reveals the outcomes of samples with a focus of 100 Bb per mL. (a) Borrelia detection after the boiling lysis experiment. (b) The impact on Borrelia focus and output when together with a centrifugation step for focus previous to boiling. (c) The impact on Borrelia rain, when together with a quick-spin step in between pattern boiling and dPCR response assembling. (d) The impact on fluorescence amplitude and rain, when subjecting the boiled pattern to proteinase Ok remedy.

The analytical sensitivity of the optimized “direct” pre-PCR technique was assessed utilizing a dilution sequence of spiked Borrelia samples within the adverse CSF matrix. The dilution sequence ranged from roughly 200 Borrelia targets per mL pattern to three targets per mL pattern. The analytical sensitivity was estimated to be roughly 10 Borrelia targets per mL pattern, when utilizing the bottom focus (0.12 copies/µL/response) calculated by the QuantaSoft software program (Fig. 4).

Determine 4
figure 4

Analysis of analytical sensitivity of the “direct” pre-PCR technique on spiked samples in adverse CSF matrix. Bb Borrelia micro organism. (a) Outcomes given in copies/µL calculated by the QuantaSoft software program. The blue squares characterize the quantification of Borrelia copies, whereas the inexperienced squares characterize the quantification of the interior optimistic management. (b) Borrelia outcomes of the dilution sequence with completely different concentrations of spiked Bb in adverse CSF matrix used for evaluation of the analytical sensitivity. One consultant output of every focus is introduced.

Comparability of “direct” pre-PCR technique to Maxwell DNA extraction technique (C)

We carried out a direct comparability of the optimised direct pre-PCR technique to a industrial DNA extraction platform, the Maxwell purification system (Promega). Bb had been spiked into both “adverse CSF matrix” or the “CSF matrix with pleocytosis” and run in parallel utilizing the optimised direct technique or the Maxwell DNA extraction technique as detailed in Supplementary C.

The examined strategies confirmed comparable outcomes relating to whole droplet counts and goal quantification. Nevertheless, the Maxwell extraction technique demonstrated a greater clustering of optimistic and adverse partitions in comparison with the direct pre-PCR technique, relating to each CSF matrix with and with out pleocytosis (Fig. 5). Moreover, the mobile elements within the CSF matrix with pleocytosis did have a transparent adverse impact on PCR amplification of each the Borrelia and the IPC dPCR assays within the direct pre-PCR technique in comparison with the Maxwell technique, as indicated by the rise in quantity of rain. The analytical sensitivity of the Maxwell technique was evaluated to be roughly 10 Borrelia targets per mL pattern, when utilizing the bottom focus (0.14 copies/µL) calculated by the QuantaSoft software program (Fig. 6). Due to these outcomes, we determined to proceed the investigations of the scientific samples with the Maxwell purification technique.

Determine 5
figure 5

Comparability of “direct” pre-PCR technique to industrial Maxwell DNA extraction technique. The blue and inexperienced dots characterize the Borrelia optimistic partitions and IPC optimistic partitions respectively, and gray dots characterize the adverse partitions. The horizontal pink traces illustrate the guide threshold setting for the info evaluation. Amplitude on X and Y-axis refers to fluorescence depth of particular person partitions. For each spike experiment Borrelia garinii pressure Lu118 was counted and diluted with both “adverse CSF matrix” or “CSF matrix with pleocytosis”. Every determine reveals the outcomes of samples with a focus of fifty Bb per mL. (a) “direct” pre-PCR technique of samples in “adverse CSF matrix”. (b) Maxwell purification of samples in “adverse CSF matrix”, (c) “direct” pre-PCR technique of samples in “CSF matrix with pleocytosis”. (d) Maxwell purification of samples in “CSF matrix with pleocytosis”.

Determine 6
figure 6

Analysis of the analytical sensitivity of the Maxwell DNA extraction technique. Bb Borrelia micro organism. (a) Outcomes given in copies/µL calculated by the QuantaSoft software program. The blue squares characterize the quantification of Borrelia copies, whereas the inexperienced squares characterize the quantification of the interior optimistic management. (b) Borrelia dPCR outcomes of the dilution sequence with completely different concentrations of Bb spiked in adverse CSF matrix used for evaluation of the analytical sensitivity. One consultant dPCR output of every focus is introduced.

Borrelia dPCR on affected person samples

The scientific validation of the Borrelia dPCR technique was carried out on fifty-nine CSF samples from sufferers examined for LNB, 9 sufferers with optimistic CSF/serum Bb-specific antibody index (AI) outcomes and fifty sufferers with adverse AI outcomes and no CSF pleocytosis, respectively. All affected person samples had been centrifuged, and DNA was extracted utilizing the Maxwell instrument, earlier than dPCR evaluation.

The period of sickness, ages, and the laboratory findings of the 9 LNB sufferers are introduced in Desk 1. One affected person pattern was optimistic for Borrelia DNA within the dPCR evaluation. Thus, the scientific sensitivity was calculated to be 11.1% (1/9) and the scientific specificity was 100% (50/50). The affected person with the optimistic Borrelia dPCR check had a brief period of signs (< 7 days) previous to lumbar puncture. The preliminary Borrelia dPCR consequence was confirmed by a repeat run on the identical pattern eluate. Each dPCR runs demonstrated very low quantities of Borrelia DNA within the affected person pattern (Fig. 7) similar to roughly 10 Bb per mL affected person CSF pattern.

Desk 1 Scientific and laboratory findings in cerebrospinal fluid (CSF) samples from 9 sufferers with Lyme neuroborreliosis.
Determine 7
figure 7

2D-dPCR output demonstrating the optimistic consequence a affected person pattern. (a) 1st dPCR run. (b) Repeat run. The blue and inexperienced dots characterize the Borrelia optimistic partitions and IPC optimistic partitions respectively, and gray dots characterize the adverse partitions. The horizontal pink traces illustrate the guide threshold setting for the info evaluation. Amplitude on X and Y-axis refers to fluorescence depth of particular person partitions. The crimson circles spotlight the Borrelia optimistic partitions. The inserts visualize enlarged optimistic partitions.



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