Melting temperature mapping method using imperfect-match linear long probes

Identifying pathogenic microorganisms as early as possible is critical for selecting the appropriate antimicrobial therapy in infected patients. We previously reported the development of the Tm mapping method for identifying a broad range of pathogenic bacteria within 3 h of blood collection. However, the Tm mapping identification requires an analytical instrument with a tube-to-tube variation of no more than 0.1 °C, so we can only use a few instruments that have such high thermal accuracy. To address the problem, we developed the improved Tm mapping method using imperfect-match linear long quenching probes (IMLL Q-probes). Using IMLL Q-probes, almost all commercially available analytical instruments can be used for the Tm mapping method. Some bacterial species cannot be narrowed down to one species, but they can at least be narrowed down to the genus level. The Tm mapping method using IMLL Q-probes is useful for deciding on antimicrobial therapy in infected patients.

the improved Tm mapping method using IMLL Q-probes can be used with almost all commercially available real-time PCR instruments.
This method consists of four major steps.First, bacterial DNA are extracted directly from a clinical sample (2 mL of a whole blood sample, etc.) as a template for PCR.Step two involves nested PCR using five bacterial universal primer sets (one primer set per tube in the second PCR); these primers can amplify almost all species of bacteria (Fig. 1b) but does not amplify fungi nor human DNA.To achieve accuracy in this PCR step, we developed a eukaryote-made thermostable DNA polymerase that is free from bacterial DNA contamination 10 .The eukaryote-made thermostable DNA polymerase is a recombinant polymerase manufactured using eukaryotic (yeast) host cells.Employing this DNA polymerase, sensitive and reliable detection of bacteria without false-positive results is feasible, thereby making it possible for PCR to identify bacterial isolates directly from patient samples.The nested PCR procedure is performed, and five (or fewer) PCR amplicons are obtained.In step three, Region 1 and 3 PCR amplicons are divided into two parts, and then a total of seven PCR amplicons are mixed with seven kinds of IMLL Q-probes (Fig. 1a-c).Specifically, Probe 1-1 is mixed in a PCR tube containing Region 1 amplicon, Probe 1-2 is mixed in a PCR tube containing Region 1 amplicon, Probe 2 is mixed in a PCR tube containing Region 2 amplicon, etc.Seven Tm values are then acquired by analyzing the seven IMLL Q-probes.Step four involves mapping the seven Tm values in two dimensions (see Fig. 1a).The plot creates a unique species-specific shape known as the Tm mapping shape.Of note: this is not a high-resolution meltingcurve (HRM) analysis, and only the Tm values are recorded.By comparing the Tm mapping shape to the shapes in the database, the bacterial isolates can be rapidly identified.

Strategies in designing IMLL Q-probes
In order for the improved Tm mapping method to work on almost all real-time PCR instruments, we designed the IMLL Q-probes.Four points are important to consider when designing IMLL Q-probes.First, strive to make it possible to obtain a wider variation range of Tm values for different species of bacteria.Second, design probes long enough (around 40-mer) to bind almost all species of bacteria, even those with many probe-target mismatches.Third, design long probes that do not have secondary structures but rather have linear structures to prevent self-quenching.Finally, design probes so as to obtain a wide variety of Tm values for each bacterial species and to identify various bacterial species.
We designed seven long probes that could bind positions with many probe-target mismatches (Supplemental Table S1).A sufficient probe length creates more hydrogen bonds than mismatches, so the IMLL Q-probe can bind many bacterial species with different base sequences.Furthermore, using the delta G value, these long probes were strictly designed to prevent a secondary structure in the probes themselves from forming.The delta G value of any self-dimers, hairpins, and heterodimers should be weaker (more positive) than − 9.0 kcal/mol.Positive numbers indicate that the actual secondary structure shown will not form at all.We named these long quenching probes imperfect-match linear long quenching probes (IMLL Q-probes).

Construction of the Tm mapping database using IMLL Q-probes
Using the mean of triplicate Tm value measurements with IMLL Q-probes, we constructed a preliminary database with the Tm mapping shapes of 68 species (Fig. 2).These species of bacteria were obtained from clinical samples and then Sanger sequenced and identified at the species level.The Tm mapping database is scalable and can be easily modified and updated.The individual Tm mapping shapes in the database show unique shapes reflecting the number and position of probe-target mismatches on IMLL Q-probe hybridizations of each bacterial species.Some Tm mapping shapes are missing data points; this is due to the fact that such Tm values cannot be obtained because the IMLL Q-probes do not bind to their target regions.To identify the bacterial isolate, the identification software program narrows the scope of its search to bacteria in the database with the same pattern as the IMLL Q-probe binding while also comparing the Tm mapping shapes.The pattern of the IMLL Q-probe binding is therefore also a characteristic of the bacteria.
Using the current protocols, the limits of identification are as follows: Escherichia coli = 5 CFU/PCR tube (250 CFU/mL), Staphylococcus aureus = 5 CFU/PCR tube (250 CFU/mL).The limits of identification were determined to be the final log 2 dilution of the template in which the Tm mapping result was correct.

Assessment of the accuracy of the Tm mapping method using IMLL Q-probes
To assess the accuracy of the Tm mapping method using IMLL Q-probes, we first performed blind tests using the 68 species of bacterial DNA registered in the database.Concealing the name of the bacteria, we tried to identify the bacterial DNA using the Rotor-Gene Q instrument (tube-to-tube variation ≤ ± 0.1 °C).Of the 68 bacterial species, 62 Tm mapping results matched the pre-sequenced bacterial DNA by Sanger sequencing, but 6 Tm mapping results were unable to be narrowed down to a single bacterial species.The Tm mapping method using IMLL Q-probes was unable to distinguish between Enterococcus faecalis and Enterococcus faecium or between Staphylococcus aureus, Staphylococcus hemolyticus, Staphylococcus hominis and Staphylococcus lugdunensis.The mean difference value was 0.242, with a range of 0.09-0.30(standard deviation = 0.08).
Next, to confirm the ability to identify bacteria depending on real-time PCR instruments, we assess the Tm mapping shape similarity among 68 species of bacteria in the database (Table 1).We previously defined the difference value as the difference between the Tm mapping shape and that observed in the database 9 .The closer the difference value is to 0, the more similar the Tm mapping shape is to the shape of the bacteria registered in the database.If the tube-to-tube variation of the real-time PCR instrument is ≤ ± 0.1 °C, then the measurement error in the difference values would be ≤ 0.28, as calculated by the difference value formula: √ (7×(±0.1) 2 ) .In the same way, if the tube-to-tube variation of the real-time PCR instrument is ≤ ± 0.2 °C, ≤ ± 0.3 °C, ≤ ± 0.4 °C, ≤ ± 0.5 °C, or ≤ ± 0.6 °C, then the measurement error in the difference values would be ≤ 0.53, ≤ 0.80, ≤ 1.06, ≤ 1 .33,and ≤ 1.59, respectively (Table 2).The tube-to-tube variation of almost all commercially available real-time PCR instruments is ≤ ± 0.5 °C, resulting in a difference value measurement error of ≤ 1.33.Based on the data obtained using the IMLL Q-probes, 60 of the 68 bacterial species in the database do not interfere with each other if the difference value measurement error is ≤ 1.33 (see Tables 1 and 2).Therefore, with almost all commercially available real-time PCR instruments, the Tm mapping method using IMLL Q-probes was able to identify at least 60 out of 68 bacteria at the species level; the remaining 8 Tm mapping results were matched at the genus level but were unable to be narrowed down to a single bacterial species.
Finally, using 18 whole blood samples collected from patients with sepsis, we compared the accuracy of the Tm mapping method using IMLL Q-probes with the LightCycler 480 instrument (tube-to-tube variation ≤ ± 0.4 °C) with that of the conventional culture method.The individual Tm mapping results using IMLL Q-probes compared with the culture or sequencing results are shown in Table 3.If the Tm mapping result did not match the culture result, we checked it again using the Sanger sequencing method (Supplementary Data).As a negative control, no bacteria were detected in healthy whole blood using either Tm mapping method or conventional culture method.Of a total of 18 Tm mapping results, 13 Tm mapping results were a match with the culture or sequencing results at the species level, but concerning Enterococcus faecalis or genus Staphylococcus, Tm mapping results were unable to be narrowed down to a single bacterial species.For example, in patient No. 16, the Tm mapping result was genus Staphylococcus (Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus hominis or Staphylococcus lugdunensis) because the difference values were < 0.28, whereas the culture result was Staphylococcus aureus.We additionally designed a short probe for specifically identifying Staphylococcus aureus (see "Methods" section).In this case, we used the short probe for Staphylococcus aureus, which only takes  www.nature.com/scientificreports/another 10 min, and identified that it was Staphylococcus aureus.In contrast, in patient No. 15, the Tm mapping result was genus Staphylococcus, whereas the culture result was coagulase-negative Staphylococcus (CNS), and the sequencing result was Staphylococcus caprae.We then used the short probe for S. aureus and found that it was not S. aureus.In addition, in patient No. 9, the culture results showed Haemophilus influenzae, Klebsiella oxytoca, and Streptococcus pneumoniae, while the Tm mapping and sequencing results showed only Haemophilus influenzae.This is because the Tm mapping method using IMLL Q-probes is able to identify only the dominant bacteria in a clinical sample.

Discussion
In the previous manuscript concerning the Tm mapping method, we mainly discussed the measurement error caused by the instrument, as the ranges of Tm values of the current Tm mapping method are < 4 °C9 .In the current Tm mapping method, over 160 species of bacteria are registered in the database within a range of only 4 °C.Therefore, it is very significant to choose an analytical instrument for the Tm mapping method that can exactly measure Tm values with less measurement error.However, there are only a few optimal real-time PCR instruments for the current Tm mapping method, which requires a tube-to-tube variation of ≤ ± 0.1 °C, such as the RotorGeneQ or LightCycler® Nano.
To address this issue, we developed the improved Tm mapping method using IMLL Q-probes.The ranges of Tm values of the IMLL Q-probes exceed 20 °C, so real-time PCR instruments with a tube-to-tube variations of ≤ ± 0.5 °C are theoretically available for use with the improved Tm mapping method using IMLL Q-probes.The tube-to-tube variations of almost all commercially available real-time PCR instruments are from ± 0.3 to ± 0.5 °C, so the improved Tm mapping method using IMLL Q-probes can be performed with almost all real-time PCR instruments.As a result, the improved Tm mapping method using IMLL Q-probes is far superior to the previous Tm mapping method in terms of versatility of instrument, but the previous Tm mapping method is superior in identification accuracy and LOI (Limit of Identification).For example, the LOI results using E. coli were 1.25 vs. 5.0 CFU/PCR tube for the previous method vs. the improved method.However, since accuracy and reproducibility of these methods depend on the temperature control ability of the real-time PCR instrument, it is difficult to say which one is better.
Chakravorty et al. reported the rapid identification of bacterial isolates using a sloppy molecular beacon (SMB) melting temperature signature technique 11 .In their study, the authors used six Tm values as signatures of bacterial isolates to identify bacterial isolates using the D value as the distance between two points in sixdimensional space.The basic concept of the SMB melting temperature signature technique is similar to that of the Tm mapping method.In the present study, we referenced their SMB technique, a type of DNA probe.Tm variations in the SMB melting temperature signature technique depend on the number and position of probetarget mismatches on SMB hybridization.Therefore, the SMB melting temperature signature technique can be used to generate a wider variation range of Tm values than the current Tm mapping method, which means that tube-to-tube variation is not a major problem for the SMB melting temperature signature technique.
We then attempted to develop an improved Tm mapping method using long DNA probes.In this venture, in order to make a long probe (around 40-mer) without allowing the formation of secondary structures in the probes themselves, we first question whether or not molecular beacon structure was indispensable.To design an imperfect-match long probe, the target position in 16S ribosomal RNA gene is limited to some extent, but there is substantial flexibility in the design because the probe uses "imperfect-match" hybridization.By the way, degenerate probes are convenient for probe-target binding, but because multiple Tm values overlap, they are not useful for bacterial species identification using Tm values as in this method.www.nature.com/scientificreports/ We designed and created many long probes using the delta G value and performed many operation checks until finally successfully choosing seven optimal long quenching probes without formation of secondary structure (i.e.without self-quenching).We named these long quenching probes the "imperfect-match linear long quenching probes (IMLL Q-probes)".The major difference between the SMB melting temperature signature technique and the improved Tm mapping method using IMLL Q-probes is that the novel Tm mapping method performs nested PCR using bacterial universal primers and the eukaryote-made thermostable DNA polymerase and then uses IMLL Q-probes, thereby making it possible for PCR to identify bacterial isolates directly from patient samples within three hours of sample collection.Without the eukaryote-made thermostable DNA polymerase, it would be difficult to identify the bacterial isolate directly from whole blood samples or diagnose the absence of bacteria in a given sample.Namely, due to the superiority of eukaryote-made thermostable DNA polymerase and nested PCR method, the improved Tm mapping method has better LOI compared to the SMB melting temperature signature technique.As for versatility of instrument, both methods would be equally good.Regarding identification accuracy, it is difficult to say which one is better because it depends not only on the probe design but on the number and type of bacteria registered in the database.In addition, since accuracy and reproducibility of these methods depend on the temperature control ability of the real-time PCR instrument, it is also difficult to say which one is better.www.nature.com/scientificreports/Tm variations in the Tm mapping method using IMLL Q-probes depend on the number and position of probe-target mismatches on IMLL Q-probes hybridization.Therefore, the variation of Tm mapping shapes also depends on the number and position of probe-target mismatches on the hybridization.Table 1 shows the number of bacterial species registered in the database with a similar shape based on the difference value, demonstrating the identification accuracy of each species of bacteria based on mutual Tm mapping shape similarity.Each difference value was calculated using the mean of triplicate Tm values already measured by the Rotor-Gene Q instrument.This similarity does not always interfere with the Tm mapping identification, but a result of around 6 bacterial species (difference value ≤ 0.28) is difficult to narrow down to a single species using any optimal instruments (see Table 2).Even when using analytical instruments with tube-to-tube variations of ≤ ± 0.1 °C, bacterial species registered in the database that cannot be narrowed down to a single species included Enterococcus faecalis vs. Enterococcus faecium and Staphylococcus aureus vs. Staphylococcus hemolyticus, Staphylococcus hominis, and Staphylococcus lugdunensis.This is because there is a limit to the diversity of target base sequences of IMLL Q-probes when they belong to the same genus.However, for the bacteria mentioned above, most commercially available analytical instruments can narrow them down to at least the genus level.Concerning genus Staphylococcus, we made a short probe for specifically identifying Staphylococcus aureus and successfully distinguished Staphylococcus aureus from CNS (see Table 3), which only took another 10 min to perform.
In conclusion, the Tm mapping method using IMLL Q-probes enables the identification of the dominant bacteria in a clinical sample within three hours of whole blood collection.Notably, almost all commercially available real-time PCR instruments can be used with the improved Tm mapping method using the IMLL Q-probes, in contrast to the current Tm mapping method.Some bacterial species cannot be narrowed down to a single species, but they can at least be narrowed down to the genus level.In such cases, tailored short probes for identifying specific bacteria can be used, which only takes another 10 min to perform.The Tm mapping method using IMLL Q-probes is particularly useful for detecting infectious diseases, such as sepsis, that require prompt treatment, and is expected to contribute to the treatment of patients with severe infections.Table 3. Individual results of identification starting from whole blood samples using a LightCycler 480 instrument (tube-to-tube variation ≤ ± 0.4 °C).Diff., difference value.✔, Matched the culture/sequencing result.-, Did not perform a comparison with the culture/sequencing result.*Nucleotide sequence data are shown in the Supplementary Data File.

Methods
The Tm mapping method using IMLL Q-probes described here is an improvement of the Tm mapping method we previously reported 9 .Therefore, it should be noted that most of the methods are the same as the previously reported Tm mapping method.

Clinical specimens
A total of 18 whole blood samples were collected from patients with sepsis at Toyama University Hospital and Nagaresugi Geriatric Hospital.In addition, a whole blood sample was collected from a single healthy volunteer at Toyama University Hospital.All procedures were performed under a protocol approved by the Ethics Committee at the University of Toyama and Nagaresugi Geriatric Hospital, and written informed consent was obtained from all patients.The methods were carried out in accordance with the approved guidelines.

Isolation of bacterial genomic DNA from whole blood
A total of 2 mL of venous blood or, as a negative control for DNA extraction, 2 mL of molecular-grade distilled water (water deionized and sterilized for molecular biology; Nacalai Tesque, Inc., Kyoto, Japan) was collected in EDTA-2K tubes (BD Biosciences Japan, Tokyo, Japan).The blood samples were then centrifuged at 100 × g for 5 min to spin down the blood cells, and the resulting supernatant fractions (1 mL) were used.The supernatants were centrifuged again at 20,000 × g for 10 min, and 950 μL of the supernatant fractions was carefully removed, taking to avoid disturbing the pellets.Next, 1 mL of molecular-grade distilled water (water deionized and sterilized for molecular biology; Nacalai Tesque, Inc.) was added to the pellets, and the mixture was gently turned upside down several times and subsequently centrifuged at 20,000 × g for 5 min.Finally, 1 mL of the supernatant fractions was again carefully removed unless the pellet was resuspended before using the DNA extraction kit.DNA was isolated from the pellets using a DNA extraction kit (QIAamp UCP Pathogen Mini Kit; Qiagen) in accordance with the supplier's instructions.Finally, bacterial DNA was eluted with 100 μL of elution buffer.

Isolation of bacterial genomic DNA from bacterial colonies
The bacterial colonies were selected with a sterile inoculating loop and suspended in 1 mL of molecular-grade distilled water (water deionized and sterilized for molecular biology; Nacalai Tesque, Inc.).The samples were subsequently centrifuged at 20,000 × g for 10 min, and 950 μL of the supernatant was carefully removed while taking care to avoid disturbing the pellets.DNA was isolated from the resulting pellets using a DNA extraction kit (QIAamp UCP Pathogen Mini Kit; Qiagen) in accordance with the supplier's instructions.Finally, bacterial DNA was eluted with 100 μL of elution buffer.

Figure 1 .
Figure 1.Workflow of the melting temperature mapping method using imperfect-match linear long quenching probes (IMLL Q-probes).(a) Workflow of the Tm mapping method using IMLL Q-probes for identifying unknown pathogenic bacteria within three hours of whole blood collection.(b) The strategy for the primer designs is shown.Nested PCR is performed using five bacterial universal primer sets, seven IMLL Q-probes are additionally used for PCR amplicons respectively, and then seven Tm values are obtained.(c) Binding sites of each IMLL Q-probes.

Figure 2 .
Figure 2. The Tm mapping shapes (IMLL Q-probes) of the 68 species of bacteria registered in the database.The horizontal line drawn in each Tm mapping shape is the average of the seven or fewer Tm values using IMLL Q-probes.

Table 1 .
Tm mapping shape similarity among 68 species of bacteria in the database.Non-zero numbers are in [bold].D, difference value.

Table 2 .
Bacterial species that are indistinguishable using various instruments.