Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 22
Issue 2
DOI:10.30825/5.ejpau.175.2019.22.2 , EJPAU 22(2), #05.
Available Online: http://www.ejpau.media.pl/volume22/issue2/art-05.html


Anna Baturo-Cieśniewska1, Giuseppe Loddi2, Janusz Prusiński3, Aleksander Łukanowski1
1 Laboratory of Phytopathology and Molecular Mycology, Department of Biology and Plant Pathology, Faculty of Agriculture and Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland
2 Consortium UNO, Faculty of Biology and Pharmacy, University of Cagliari, Oristano, Italy
3 Department of Agrotechnology, Faculty of Agriculture and Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland



Several protocols of DNA extraction from the soil with commercial kits and CTAB based method were tested to develop relatively easy procedure of reliable molecular detection of B. japonicum introduced with coated soybean seeds.
Method of DNA extraction clearly influences the quality and quantity of DNA and thus directly affects the results of PCR (Polymerase Chain Reaction) and qPCR (quantitative Polymerase Chain Reaction) assays and the possibility of bacteria identification. Obtaining reliable results in PCR does not guarantee the same results in qPCR with SYBR Green. The use of DNA extraction commercial kits and successful DNA extraction may in some cases lead to inability to detect bacteria, although it is present in the sample. To avoid such problems, the extraction methods should be selected and, if necessary, modified individually for different soil samples. In this study, DNA extraction with Genomic MINI AX Soil Spin showed to be the most appropriate method for obtaining reliable results in both PCR and real-time PCR. However, for real-time PCR, strong dilution of DNA and the addition of reagents that relieve inhibition of amplification were necessary.

Key words: Bradyrhizobium japonicum, DNA extraction, PCR inhibitors, real-time PCR, soil.


Soil biodiversity represents a huge underground world containing archaea, bacteria, fungi, nematodes, insects and earthworms. These organisms interact with each other and affect the functioning of the soil ecosystem [42]. They play fundamental role in various biogeochemical processes, they are responsible for the cycling of organic compounds, also contribute to plant nutrition and soil fertility [10, 17, 25]. DNA isolation and obtaining good quality extracts of nucleic acids is the basic and probably the most important step in molecular analyses of microbial community, which affects the reliability of final results [23, 40]. The purity of the DNA extracted from soil is often unsatisfactory, particularly in soils rich in humic compounds [6]. Their physico-chemical similarity with nucleic acids cause that they are extracted together with DNA [45]. Humic acids (HA) interact with the template DNA and the Taq DNA polymerase even at low concentrations and prevent the enzymatic reaction such as qPCR [37, 38]. Also proteins as impurities of soil DNA extracts can cause inhibition in the PCR [19]. Co-extracted substances that inhibit PCR can lead to inaccurate results and subsequent misinterpretation about a species’ status in the tested system. The consequences of partial or full inhibition are a decrease in assay sensitivity and the increased potential for false negatives [21]. Despite the fact that many commercial tests were compared and many studies [7, 8, 10, 11, 15, 24, 27, 31] have been conducted to develop the most appropriate protocols for extracting microbial DNA and different isolation and purification methods to inactivate and remove PCR inhibitors were compared for soil samples, the problem still exists.

In the cultivation of legumes, nodulation bacteria typical for different plant species play a special role for producing high yield as well as for keeping the soil fertile for the succeeding crops [14]. To enrich the soil in Bradyrhizobium japonicum that coexists with soybean, inoculation of soybean seeds is widely applied [3, 34]. However, the effectiveness of preparations containing this bacterium can be varied. Alves et al. [3] noted that several efficient imported Bradyrhizobium strains can be unable to compete with native soil micro-flora and other previously-introduced Bradyrhizobium strains. Therefore, it is important to verify that the inoculation results in B. japonicum development in the root area. As our preliminary unpublished studies showed, there is no problem with the molecular identification of the level of root colonization by B. japonicum. Soil analysis turned out to be problematic, the results were confusing, despite of the DNA extraction with recommended commercial kits. Their producers claim that DNA can be used directly for further analysis such as enzymatic reactions with real-time detection and other procedures requiring high-quality of nucleic acids.

The aim of the study was to test several methods of DNA extraction basing on kits provided usually by manufacturers for a few samples and to propose a procedure that enables reliable detection of B. japonicum with PCR and qPCR in soybean rhizosphere soil.


Taking soil samples
Rhizosphere soil samples were taken in reproductive soybean growth stage R2 (Full flowering) when nodulation was at maximum from plants cultivated in experimental station in Mochełek, Poland (53°13'N, 17°51'E) belonging to UTP University of Science and Technology in Bydgoszcz, Poland. Soil was classified as luvisol, very good rye complex and soil valuation class IVa; pH in KCl was 6.0.

Two samples A and B were selected for the study. They originated from experimental fields, where B. japonicum was introduced to the soil with seeds coated with Soybean Inoculants Hi Stick (BASF), that gave a high probability of bacterial DNA occurrence in rhizosphere and roots. Samples were collected to sterile Falcon tubes 50 ml and freeze-dried (CoolSAFE – Scanvac).

DNA extraction
DNA was extracted with 5 commercial kits: GeneMATRIX Soil DNA Purification Kit (EURx) (Method 1), Syngen Soil DNA Mini Kit (Syngen Biotech) (Method 2), Genomic MINI AX Soil Spin (A&A Biotechnology) (Method 3), PowerSoil DNA Isolation Kit (Mo BIO /Qiagen) (Method 4) and GeneMATRIX Environmental DNA & RNA Purification Kit (EURx) (Method 5) according to manufacturer’s protocols. Additionally, DNA was isolated from 500 mg of soil samples with traditional method i.e. according to modified Doyle and Doyle (DD) protocol [9] in 900 µl of extraction buffer that, apart from CTAB 5.0%, EDTA 0.5 M, NaCl 5.0 M, Tris-HCl (pH 8.0) 1.0 M and b-mercaptoethanol, contained PVP 2.0% (Method  6a) or 0.03 g PVPP (Method 6b) per sample. In further steps phenol, isoamyl alcohol and chloroform (25:24:1, v/v) were applied for DNA cleaning. The DNA was precipitated with ethyl alcohol 96% and suspended in ddH2O. Before DNA extraction with DD method, four soil samples (Method 6c) were homogenized with sterile glass beads 2-mm diameter in MagnaLyser (Roche) at 5000 rpm for 90 s in order to detach the cells from soil matrix and to obtain the cell lysis. Finally, 32 samples of DNA were obtained: A and B not cleaned (-) and A’ and B’ additionally cleaned (+) with Anty-inhibitor Kit (A&A Biotechnology). Details on extracted DNA samples are shown in Table 1.

Table 1. Details on DNA extraction methods and DNA quantity and quality
No Method of DNA extraction Soil sample [mg] Extrac-
DNA [µl]
tor Kit
DNA yield
260 / 280 nm
DNA purity 260 / 230 nm
S1 A1 – GeneMATRIX Soil DNA Purification Kit (EURx) (Method 1) 200 100 - 48.5 1.78 1.17
(Method 1)
200 100 + 32 2.14 0.87
S3 B – GeneMATRIX Soil DNA Purification Kit (EURx) (Method 1) 200 100 - 36.5 1.76 1.06
(Method 1)
200 100 + 29 2.16 0.59
S5 A – Syngen Soil DNA Mini Kit (Syngen Biotech) (Method 2) 500 100 - 35 1.48 0.81
S6 A – Syngen Soil DNA Mini Kit (Syngen Biotech) ) (Method 2) 500 100 + 27.8 1.78 0.82
S7 B – Syngen Soil DNA Mini Kit (Syngen Biotech) ) (Method 2) 500 100 - 18 1.49 0.81
S8 B – Syngen Soil DNA Mini Kit (Syngen Biotech) ) (Method 2) 500 100 + 22.5 1.72 0.78
S9 A – Genomic MINI AX Soil Spin (A&A Biotechnology) ) (Method 3) 500 150 - 1.9 1.40 0.7
S10 A – Genomic MINI AX Soil Spin (A&A Biotechnology) (Method 3) 500 150 + 1.7 1.98 0.49
S11 B – Genomic MINI AX Soil Spin (A&A Biotechnology) (Method 3) 500 150 - 1.54 1.40 0.7
S12 B – Genomic MINI AX Soil Spin (A&A Biotechnology) (Method 3) 500 150 + 1.43 2.10 0.48
S13 A – PowerSoil DNA Isolation Kit (Mo BIO /Qiagen) (Method 4) 250 100 - 15 1.72 0.9
S14 A – PowerSoil DNA Isolation Kit (Mo BIO /Qiagen) (Method 4) 250 100 + 15 5.20 0.43
S15 B – PowerSoil DNA Isolation Kit (Mo BIO/Qiagen) (Method 4) 250 100 - 13 1.53 0.88
S16 B – PowerSoil DNA Isolation Kit (Mo BIO /Qiagen) (Method 4) 250 100 + 13 2.25 0.31
S17 A – GeneMATRIX Environmental DNA & RNA Purification Kit (EURx) (Method 5) 100 40 - 43 1.73 1.05
S18 A – GeneMATRIX Environmental DNA & RNA Purification Kit (EURx) (Method 5) 100 40 + 40 2.05 0.56
S19 B – GeneMATRIX Environmental DNA & RNA Purification Kit (EURx) (Method 5) 100 40 - 29 1.71 1.28
S20 B – GeneMATRIX Environmental DNA & RNA Purification Kit (EURx) (Method 5) 100 40 + 37 1.93 0.53
S21 A – Doyle and Doyle method with PVP K 30 (2%) DD (Method 6a) 500 50 - 95 1.42 0.80
S22 A – Doyle and Doyle method with PVP K 30 (2%) (Method 6a) 500 50 + 69 1.55 0.76
S23 A – Doyle and Doyle method with PVPP (Method 6b) 500 50 - 140 1.44 0.97
S24 A – Doyle and Doyle method with PVPP (Method 6b) 500 50 + 93 1.49 0.76
S25 B – Doyle and Doyle method with PVP K 30 (2%) (Method 6a) 500 50 - 90 1.43 0.72
S26 B – Doyle and Doyle method with PVP K 30 (2%) (Method 6a) 500 50 + 79 1.48 0.75
S27 B – Doyle and Doyle method with PVPP (Method 6b) 500 50 - 100 1.43 0.72
S28 B – Doyle and Doyle method with PVPP  (Method 6b) 500 50 + 80 1.50 0.75
S29 A – Doyle and Doyle method with PVP K 30 (2%) + glass beads (Method 6c) 500 100 - 115 1.48 0.80
S30 A – Doyle and Doyle method with PVP K 30 (2%) + glass beads (Method 6c) 500 100 + 110 1.54 0.74
S31 B – Doyle and Doyle method with PVP K 30 (2%) + glass beads (Method 6c) 500 100 - 110 1.48 0.82
S32 B – Doyle and Doyle method with PVP K 30 (2%) + glass beads (Method 6c) 500 100 + 100 1.58 0.77
1 A and B – codes of soil samples

DNA quantity and quality
Concentrations of DNA in all samples were measured with QuantiFluor dsDNA on Quantus Fluorometer (Promega) according to the manufacturer’s protocol. DNA purity was determined spectrophotometrically by measuring the absorbances at 260 vs 280 nm and 260 nm vs 230 nm (NanoDrop1000, Thermo Scientific).

DNA quality was assessed by 0.9% gel (Agarose F.P. DNA, Pronadisa) electrophoresis in 1 × TBE buffer stained with SimplySafe (EURx). For all samples 8.0 µl of DNA with 1.5 µl of loading buffer were applied to the gel wells. The results were scanned into a computer imaging file with a gel documentation system with a digital camera (INTAS).  

Reference isolate preparation
As a standard (St) in determination of DNA quality in electrophoresis and as a positive control (PC) in all PCR and qPCR analyses, reference isolate of B. japonicum (NCCB 47038) was used. DNA was extracted with Genomic MINI AX Bacteria (SPIN) (A&A Biotechnology), commonly and successfully used for the isolation of bacterial DNA in our laboratory, from 3 ml of bacterial suspension originated from a colony grown for 3 days in 100 ml of Yeast Mannitol Broth (YMB) pH 6.8 on a rotary shaker (150 rpm) at 28°C.

Detection of B. japonicum with PCR
The quality of DNA was estimated also in PCR assay for B. japonicum presence in the samples. Working solutions of DNA were adjusted to 20 and 10 ng·µl-1 with the exception of samples where DNA concentration was lower. PCRs were conducted in Mastercycler Ep Gradient (Eppendorf) in a volume of 12.5 µl per sample. Each sample contained 2xPCR MixPlus (A&A Biotechnology), 0.75 µl (0.6 pM·µl-1) of each primers nodZ-A-F (5'-GGTTTGGCGACTGTCTGTGGTC-3') and nodZ-A-R (5'-TTCCACCATGTTGGAAAGAATGGTCC-3') [13], 2.5 µl of DNA and 2.25 µl ddH20 or 2 µl ddH20 and 0.25 µl Anty-inhibitor PCR (DNA Gdańsk). For low concentrated samples PCR mastermix was prepared without water and volume of DNA was 4.75 µl. The reaction conditions were as follows: 95°C for 3 min, 40 cycles (95°C for 1 min, 58°C for 1 min, 72°C for 30 s) and final extension 72°C for 5 min. Assay was performed in three independent replications to determine the presence/absence of the expected 228 bp product. The results were verified on a 1.4% agarose gel (Agarose F.P. DNA, Pronadisa) in 1 × TBE buffer stained with SimplySafe (EURx).

Detection of B. japonicum with real-time PCR
For qPCR analyses 7 samples (S9, S11, S21, S22, S24, S26 and S28) were selected, for which a single and clear expected product in PCR was obtained.

QPCR amplifications were carried out in LightCycler 480 II (Roche) in three replications for all samples. The reaction mixture consisted of 5 μl of 2 × concentrated LightCycler 480 SYBR Green I Master (Roche), 0.25 µl of each primer (10 pM·µl-1 ) nodZ-A-F /nodZ-A-R, 4.5 µl of DNA and in some cases 0.2 µl of Anty-inhibitor PCR (DNA Gdańsk). Before analysis of soil samples, primer annealing temperature was optimized with a DNA of PC 10 ng·µl-1 dilution. Finally the reaction conditions were as follows: 95°C for 10 min, followed by 45 cycles of 95°C for 10 s, 64°C for 20 s, and 72°C for 30 s. To verify the specificity of the reaction, a melting curve analyses were carried out under the following conditions: 95°C for 5 s, 65°C for 1 min, 95°C (with continuous fluorescence measurement in the range of 65°C to 95°C) and 40°C for 30 s. As a positive control (PC), a DNA isolated from the reference isolate was used in an amount of 4.5 ng per sample. The negative control (NC) reaction mixture contained water instead of DNA.

For the first test, DNA of chosen 7 samples was diluted to 10 ng·µl-1 with the exception of S9 and S11 that were directly taken to analysis. In the second test, Anty-inhibitor PCR (DNA Gdańsk) was added to reaction mixture with DNA prepared as for the first test. For further tests, DNA concentrated at 10 ng·µl-1 was additionally diluted 5 × or diluted 5 × and Anty-inhibitor PCR was added to the reaction mixture. Based on the results of preliminary experiments, only S9 and S11 diluted to 0.2 ng·µl-1 and with Anty-inhibitor PCR (DNA Gdańsk) were used in the final test. In all tests the presence of the expected product, fluorescence level, Ct value and melting curve were analyzed.


DNA quantity and quality
The yield of DNA extracted with different methods varied and ranged from 1.43 to 140 ng · µl-1. The lowest DNA concentration was obtained for Method 3 and the highest when DNA was extracted according to DD protocol, on average, 1.64 and 98.42 ng·µl-1, respectively. In both cases 500 mg of soil sample were taken to analyzes but DNA was diluted in 150 µl (Method  3) or in 50–100 µl (Method 6) (Tab. 1).

The brown color of extracted DNA and its consistency, resembling a gel, was observed in many samples. Brown dye was visible especially for samples in DD protocol. Application of Anty-inhibitor Kit resulted in improved consistency and partial discoloration, but also in reduction of DNA content in the most samples.

The value of DNA purity determined spectrophotometrically in 260 vs 280 nm ranged from 1.40 to 2.25. Values between 1.8–2.0 were found only for S10 (Method 3) and S20 (Method 5), 1.98 and 1.93, respectively. In the case of S1, S3, S6, S8, S13, S17 and S19 this parameter was between 1.7 and 1.8. The lowest values were noted for samples extracted with DD method and ranged from 1.42 (S21) to 1.58 (S32). In all cases, the use of Anty-inhibitor Kit resulted in and an increase of the ratio of absorbance at 260 nm and 280 nm. There were no DNA samples where purity measured in 260 vs 230 nm was in expected value range of 2.0–2.2. Mean value of this parameter was 0.77 and ranged from 0.31 (S16) to 1.28 (S19) (Tab. 1).

Electrophoresis showed that all DNA samples were more or less degraded (Fig. 1). In Method 6c material disruption with glass beads resulted in more intensive DNA degradation / fragmentation compared to Method 6a and 6b.

Fig. 1. Quality of 32 DNA samples extracted with different method listed in the Table 1, assessed by electrophoresis. St – standard, DNA of B. japonicum (NCCB 47038)

Detection of B. japonicum with PCR
PCR assay for B. japonicum revealed the presence of a single 228 bp product for samples where mechanical disruption during DNA extraction was not applied i.e. S9, S11 (Method 3) and S21, S22, S24-S28 (Method 6a, 6b). The DD extraction method resulted in satisfactory PCR results in most cases. No non-specific products were amplified here in contrast to results with DNA extracted with kits, with the exception of the Method no. 3 where Anty-Inhibitor Kit was not applied (Fig. 2a, 2b). For samples where DNA was extracted using commercial kits with bead beating tubes (Methods 1, 2, 4 and 5) and in method 6c with glass beads no expected product or additional non-specific products were observed. Purification of DNA with an Anty-Inhibitor Kit did not improve its quality, and in some cases (S10 and S12) had a negative effect on the course of the PCR that resulted in additional bands. Dilution of DNA and the addition of Anty-inhibitor PCR did not affect the PCR assay result.

Fig. 2. PCR products of 1–16 (a) and 17–32 (b) DNA samples with nodZ-A-F /nodZ-A-R primers. M – ladder 100 bp, PC – positive control, DNA of B. japonicum (NCCB 47038), NC – negative control

Detection of B. japonicum with real-time PCR
In the first real-time PCR test of 7 samples the amplification curve suggesting the product presence was observed only for S11 (Fig. 3). Its fluorescence level was much weaker (≈12) than for PC (≈20). In addition, the analysis of melting curve showed a slight difference between the melting point of the product S11 and PC. With Anty-inhibitor PCR, products were obtained for all 7 samples. The best results were found for S9 and S11. The fluorescence level of S11 increased compared to the first test. The analysis of S9 and S11 melting curves showed the presence of single products, but curves slightly differed from the PC. Although the products were also obtained for other samples, melting analysis showed abnormalities and the lack of reaction specificity (Fig. 4). In further tests with samples additionally diluted 5 times and with Anty-inhibitor PCR, again the best results were obtained for S9 and S11. Dilution alone resulted in a higher level of fluorescence than the addition of an Anty-inhibitor PCR to the undiluted sample. Ct value was slightly higher than with the addition of Anty-inhibitor PCR. Dilution of the samples and simultaneous addition of Anty-inhibitor PCR had a comparable effect to the dilution itself. Final test showed that addition of Anty-inhibitor PCR and sample dilution to 0.2 ng·µl-1 led to high level of fluorescence of the analyzed samples comparable to those obtained for PC, as well as high reaction specificity with melting temperature of PCR products ≈87°C (Fig. 5 and Fig. 6). Despite a significant dilution of the samples, Ct values of corresponding PCR products were not increased compared to previous tests, where higher concentrated DNA was used.

Fig. 3. QPCR analysis of 7 chosen samples diluted to 10 ng·µl-1. PC – positive control, NC – negative control

Fig. 4. Melting curves for S21, S22, S24, S26 and S28 samples diluted to 10 ng·µl-1 with Anty-inhibitor PCR and positive control (PC)

Fig. 5. QPCR analysis of S9 and S11 diluted to 0.2 ng·µl-1  and Anty-inhibitor PCR. PC – positive control, NC – negative control

Fig. 6. Melting curves for S9 and S11 diluted to 0.2 ng·µl-1  and Anty-inhibitor PCR. PC – positive control, NC – negative control


Our research showed that the method of DNA extraction clearly influenced the quality and quantity of DNA that directly affected the results of PCR assays and the possibility of reliable identification B. japonicum in soil samples. According to Tanase et al. [40] despite the development of molecular protocols for microbial DNA isolation, there are still many drawbacks dependent of samples composition, and even the commercially available genomic isolation kits have significant limitations especially for soil samples. Assurances of commercial kit manufacturers, that they enable obtaining high-quality DNA allowing for successful PCR amplification, should be treated with caution, because despite the use producer's protocols, there is the possibility of obtaining false negative results. The yield of DNA extracted with different methods varied considerably, which was mainly affected by the extraction procedure and reagents. This suggests the fact that for samples where, according to manufacturers' recommendations, the soil amount taken to extraction was lower or volume of elution buffer was lower, the higher DNA yield was not always found.

Although the DNA quality was generally poor, a product of expected size was amplified for some samples in PCR assay. Also, low concentration of DNA extracted with Method  3 was not an obstacle in obtaining the amplification product in PCR as well as real-time PCR, even for samples where the use of the Anty-inhibitor Kit (A&A Biotechnology) after DNA extraction ultimately resulted in its additional yield decrease, which probably caused its partial deposition on filtration particles.

DNA extracted with DD method seems to be suitable for reliable PCR analysis in contrast to, real-time PCR, despite the high DNA extraction efficiency. This indicates that the reliability of the results is more affected by the DNA quality related to the isolation method and reagents different for both assays than the DNA concentration. Consistency, the brown dye of DNA and 260/230 ratios of samples extracted with DD method suggested the presence (among others) of carbohydrates, that can inhibit PCR. Sajib et al. [30] reduced spongy/gel-like carbohydrate from plant DNA by collecting aqueous phase collected leaving the soft spongy pellet after final centrifugation, what is worth testing in DNA isolation from problematic samples using the DD method. Besides, probably inhibitors of PCR reactions such as humic compounds have not been completely removed from most of our samples and have disturbed the reaction. Humic substances are a mixture of complex polyphenolics produced during the decomposition of organic matter [1, 18], therefore, their presence and proteins or other contaminants may affect the 260/230 nm (DNA/HA), and 260/280 nm (DNA/protein) readings, which are determinants of DNA purity [ 16, 28, 35, 41]. For most of our samples we obtained a result showing their contamination with substances that absorb strongly at 230 or near 280 nm. PCR inhibition is the most common cause of PCR failure when adequate copies of DNA are present. Even slight inhibition of a real-time PCR may result in underestimation of DNA concentration or no detection at all [21]. A wide range of additives can be used to solubilize contaminants. In DD method we applied CTAB and PVP or PVPP. PVPP removes HA with phenolic groups from crude DNA extracts via hydrogen bounding and formation of PVPP-phenolic complexes [39]. Also a number of researchers recommend the use of PVP with molecular weight of 10,000 at 2% or 2.5% (w/v) to address the problem of phenolics [5, 29, 44]. Regardless of whether PVP or PVPP was used we obtained DNA of similar and poor quality in all samples. These chemicals can only partially remove humic compounds and even might be unreliable for the removal of inhibitors from a variety of soils [4, 12, 45]. Miao et al. [22] found that CTAB-based method is not efficient for removing contamination (e.g., carbohydrates, phenols, peptides, and aromatic compounds) from humus-rich soil samples. Despite the above, DNA amplification in our PCR assay was possible, but in qPCR all samples were problematic. We showed that the fact of obtaining reliable results in PCR does not guarantee obtaining the same result in qPCR. In PCR assays, a DNA product of expected size was obtained for templates that resulted in no product or a product that could not be the basis for reliable quantitative analyzes in qPCR. The explanation may be that humic substances cause fluorescence inhibition in real-time polymerase chain reaction and they quench fluorescence of different dyes including SYBR Green I [33, 47]. According to Schrader et al. [32], increased background fluorescence in qPCR assays represents an additional mechanism of action for PCR inhibitors that leads to decreased sensitivity. In addition, inconsistent results may have been caused by reagents, in principle different for PCR and qPCR.

Another cause of problems in the PCR and qPCR for most samples could be DNA fragmentation (degradation). In the few samples where the mechanical sample disruption did not precede the extraction of DNA, a single band (expected product) in PCR was obtained. The use of homogenization with glass beads probably disrupted the correct amplification in PCR assay. It is consistent with Tanase et al. [40] observations. Also Robe et al. [26] based on literature found that in general, physical methods shown efficient for disruption of cells and spores but may often result in significant DNA damage.

Purification of the obtained DNA contaminated with inhibitors is one of the methods to improve its quality, which directly affects PCR assays. In the case of our samples cleaning of extracted DNA with an Anty-inhibitor Kit did not bring expected results. Matheson et al. [20] showed that the use of size exclusion chromatography removed the majority of humic substances to allow successful amplification, however, such treatments usually are not carried out. Kreader [18] concludes that because purification is time consuming and increases costs of sample preparation, as well as to the loss of target nucleic acids, a more satisfying approach to the problem of PCR inhibition is to relieve interference rather than attempt to remove all of the offending substances. Among various additives that have been included in PCR to relieve inhibition is bovine serum albumin (BSA) [2, 18]. According to the producer, the Anti- inhibitor PCR, which positively influenced the results in qPCR, contains a specially selected mixture of alkaline proteins that counteract various types of substances that inhibit the PCR reaction. The beneficial effect of such proteins on qPCR with SYBR Green was noted by Wang et al. [43]. They observed reduction in the variability of peak areas and Tm values. In our research, Anti-inhibitor PCR allowed to obtain a product for some samples, however, melting curve analysis revealed abnormalities. Additionally, strong dilution of the samples had a significantly positive effect on qPCR results. Similarly, Niemi et al. [24] found that undiluted extract sometimes caused inhibition. DNA extracts obtained with method 3 were much less concentrated than DNA samples extracted using other protocols. That could be one of the reasons for obtaining the product in qPCR for S9 in the first tests, even without the addition of Anti-PCR inhibitor.

We extracted DNA with methods that are universal for extraction of DNA from different organisms present in the soil. Similarly to Niemi et al. [24], we noted significant differences in PCR results with DNA isolated in different ways and inhibition of the reaction despite the application of commercial DNA purification methods. Dineen et al. [8] suggest that commercially available extraction kits can be used to extract PCR-quality DNA from bacterial spores in soil, but the selection of an appropriate extraction kit should depend on the characteristics of the soil sample and the intended downstream application. Also target of analysis is important. According to Starke et al. [36] combination of different extraction procedures and primer sets as well may affects the results.

In our study the best results of B. japonicum identification both in PCR and qPCR were obtained for DNA extracted only with one of commercial kits. Identification of this bacteria by qPCR turned out to be more complicated than in PCR and was reliable only after diluting the samples and adding reagent containing alkaline proteins. As our preliminary (not published) researches have shown, such a procedure unfortunately does not always solve the problems and it may be helpful to use qPCR reagents for troublesome DNA templates. It is worth remembering that the success of analyses can be influenced by the diversity of soil samples, their composition and varied content of reaction inhibitors [40]. A DNA isolation method suitable for specific samples that allows achieving successful results of molecular analysis may not be suitable for others. Differences in the physicochemical properties of soil samples require optimization of DNA extraction techniques for each sample separately [46]. According to Satyanarayana et al. [31] effective purification of nucleic acids from all soils probably needs several different purification strategies.


  1. The DNA extraction method from the soil directly affects the reliability of the molecular identification of microorganisms in soil samples. The use of most kits recommended for DNA extraction from the soil in our studies did not result in reliable both PCR and qPCR identification of B. japonicum.
  2. Modified Doyle and Doyle method of DNA extraction resulted in satisfactory amplification of expected product proving the presence of B. japonicum without non-specific products in the soil samples in PCR assay, however it did not work for qPCR analysis.
  3. DNA extraction with Genomic MINI AX Soil Spin (A & A Biotechnology) turn to be the most the appropriate method for obtaining reliable results in both PCR and real-time PCR.
  4. DNA extraction methods should be selected and, if necessary, modified individually for different soil samples. Strong dilution of DNA samples and the addition of alkaline proteins can be helpful in avoiding false negative results in molecular analyzes. Real-time PCR analysis aimed at identifying B. japonicum showed that the satisfactory result was obtained for heavily diluted DNA with reagents that relieve inhibition of amplification.
  5. The analyzes should be considered preliminary due to the small number of tested samples, however, they show that based on the demo kits the selection of the appropriate method of DNA extraction in practice, which then allows to obtain reliable results, is possible.


This work was partially supported by HOR [grant number 103/OB3/3.6.2/2017].


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Received: 21.09.2019
Reviewed: 11.05.2019
Accepted: 10.06.2019

Anna Baturo-Cieśniewska
Laboratory of Phytopathology and Molecular Mycology, Department of Biology and Plant Pathology, Faculty of Agriculture and Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland
Kordeckiego St. 20
85-225 Bydgoszcz
email: baturo-a@utp.edu.pl

Giuseppe Loddi
Consortium UNO, Faculty of Biology and Pharmacy, University of Cagliari, Oristano, Italy
Carmine St.
09170 Oristano

Janusz Prusiński
Department of Agrotechnology, Faculty of Agriculture and Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland
Kordeckiego St. 20
85-225 Bydgoszcz
email: janusz.prusinski@utp.edu.pl

Aleksander Łukanowski
Laboratory of Phytopathology and Molecular Mycology, Department of Biology and Plant Pathology, Faculty of Agriculture and Biotechnology, UTP University of Science and Technology, Bydgoszcz, Poland
Kordeckiego St. 20
85-225 Bydgoszcz

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