The Extraction and Recovery Efficiency of Pure DNA for Different Types of Swabs

0

 

Abstract

The extraction and recovery efficiency of swabs used to collect evidence at crime scenes is relatively low (typically <50%) for bacterial spores and body fluids. Cell‐free deoxyribonucleic acid (DNA) is an interesting alternative compared to whole cells as a source for forensic analysis, but extraction and recovery from swabs has not been tested before using pure DNA. In this study cotton, foam, nylon flocked, polyester and rayon swabs are investigated in order to collect pure DNA isolated from saliva samples. The morphology and absorption capacity of swabs is studied. Extraction and recovery efficiencies are determined and compared to the maximum theoretical efficiency. The results indicate that a substantial part of DNA is not extracted from the swab and some types of swab seem to bind effectively with DNA. The efficiency of the different types of swab never exceeds 50%. The nylon flocked 4N6FLOQSwab used for buccal sampling performs the best.

Using a swab to collect samples is a common method to recover biological material from a crime scene, as well as for obtaining reference samples, such as buccal swabs 1. The different types of swabs fall into three main categories of design. The wound swab is made up of either many fibers or one long fiber wound around a shaft. The flocked swab has small fibers glued onto the shaft, and the pad swab contains a sleeve of foam (or other porous material) attached to the shaft 1. In this study cotton, foam, nylon flocked, polyester and rayon swabs are investigated.

Cotton swabs are widely available and routinely used. However, cotton swabs may leave cotton fibers or other impurities in the reaction mixture, which may negatively influence the polymerase chain reaction (PCR; e.g., by inhibition) 2. Another drawback of tightly wound swabs, such as cotton or rayon, can be their low retrieval and release performance 3.

Swabs made from (macro)foam have more open structure when compared to the polyester, rayon and cotton swabs 2. It has been suggested that foam swabs, due to the flexible nature of the material, can penetrate into porous substrates, e.g., wooden surfaces 1.

Nylon flocked swabs consist of short nylon fiber strands attached to a (plastic) shaft. The hydrophilic open fiber morphology is specially designed to improve sampling, since this structure should result in efficient collection and release of the sample. The swabs are suitable for the sampling of saliva, blood and skin epithelial cells from various substrates. Nylon flocked swabs show a better sample release and no sample entrapment as is the case with conventional cotton swabs 4. However, flocked swabs (and layered cotton) might leave swab material on the surface, especially on rough textures 1.

Rayon, also known as viscose, is a spun cellulose fiber, which is made from wood pulp and is mainly designed for the recovery of microorganisms. The texture and absorbance characteristics are similar to cotton 56.

Polyester, also known as dacron, is a nonchemisorbing synthetic polymer fiber, with high collection and release characteristics. Swabs with knitted polyester tips are widely used for cleaning purposes 56.

The performance of a sampling swab can be expressed in terms of its extraction and recovery efficiency. The extraction efficiency of a swab is defined as the material transfer effectiveness from the collection tool to the extraction solution 27. The recovery efficiency is defined as the overall transfer effectiveness from a sampled surface to the extraction solution 27.

Cotton 278, (macro)foam 28, nylon flocked 79, polyester (dacron) 28 and rayon swabs 2810 have previously been evaluated for the sampling of bacterial spores 2710 expressed in colony forming units (cfu). Typical extraction buffer volumes for these experiments have been in the order of several milliliters 2710.

For forensic (human biological) applications cotton 131118, foam 1, nylon flocked 134131516, polyester 114, and rayon (viscose) 116 swabs have been tested with epithelial 117, saliva 131416 and vaginal samples 4 and have been expressed in terms of the number of alleles present in the DNA‐profile (4111318), the DNA quantity/yield (ng), the concentration (ng/μL) or percentage 1341318. The various swabs have been ranked by Verdon et al. 1 according to their combined collection and extraction efficiency. Typical extraction volumes for forensic applications have been in the order of microliters 1341118. Only a few experiments have been performed to determine the extraction efficiency, whereas a swab may have high collection properties, but bad performance upon release of the sample 1151719. Although the absorption capacity can have a substantial influence on the DNA extraction and recovery efficiency of a swab 1, none of the reported investigations has taken this parameter into account for various types of swabs.

The presence of circulating cell‐free DNA has already been reported in the medical field (e.g., oncology), but has recently also become of interest to forensic scientists, since DNA profiles can be generated from cell‐free DNA 20. This DNA can be present as extracellular DNA, but can also originate from cell membrane rupture due to osmotic lysis caused by soaking off the stain with water 21. Cell free nucleic acids ranging from 0 to 7 ng of recoverable DNA are found in cell free sweat samples of 150 μL, with an average of 11.5 ng/mL. In a study by Quinones et al. 22 8 out of the 10 samples showed a partial or full DNA profile (even with measured quantities below 1 ng). In another study, 100 forensic case samples were investigated and cell free DNA turned out to be present in 90% of the samples from different origins (e.g., blood, saliva, vomit and contact traces) 21.

It was previously reported that for forensic samples 20–76% of the collected DNA is lost during the extraction phase 23. To investigate this problem, first the absorption capacity of a number of different swab types was determined. This absorption capacity was related to swab morphology based on scanning electron microscope images. It is worth mentioning that in forensic case research so‐called spin baskets are commonly used for the extraction and purification of DNA from cells. These baskets are developed and recommended for use in combination with a lysis step. Such a step is not required in our study since the samples consist of pure DNA. Furthermore, the basket protocols contain an intensive set of washing and purification steps, which are not required for pure DNA and may in fact be responsible for extra loss of DNA. Although other methods, such as sonication or minimal agitation, may perform equally well, Rose et al. 2 concluded that vortexing swabs for about 2 min is more efficient than sonication or minimal agitation. For this reason we have chosen this convenient method in this study. A maximum theoretical extraction (MTE) was calculated based on the assumption that the DNA sample should be homogeneously distributed over the liquid phases on the swab and in the extraction vial. The MTE was subsequently used for the evaluation of potential loss mechanisms.

Materials and Methods

Swabs

Table 1 shows an overview of the five different swab materials that were evaluated: cotton, foam, rayon, polyester, and nylon flocked. These swabs were developed for biological (e.g., collection of bacteriologic specimens), cleaning or forensic purposes. Swabs #6‐#11 were provided by ITW Texwipe (Hoofddorp, NL), swabs #12 and #13 were provided by Copan (Italia, Brescia, Italy) and swab #14 was provided by bioTRADING Benelux B.V. Mijdrecht, NL.

Table 1. Overview of the swabs used in this research. B, biological; C, cleaning; F, forensics; PP, polypropylene; PS, polystyrene
Name Manufacturer Type Shaft Order number Purpose
#1 Invasive sterile EUROTUBO® collection swab Deltalab Cotton Wood 300250 B
#2 eSwab™ Copan Nylon flocked Plastic 490C B
#3 Transwab® MWE Rayon Plastic MW170 B
#4 Dryswab™ MWE Rayon Plastic MW102 F
#5 Transport swab APTACA Rayon Plastic 301/SG B
#6 Nylon MicroSwab™ Texwipe® Nylon PP TX730 C
#7 Absorbond® with long handle Texwipe® Polyester PP TX762 C
#8 Medium CleanFoam® swab with long handle Texwipe® Foam (Polyurethane) PP TX740B C
#9 Polyester Honeycomb Swab Texwipe® Polyester PP TX802 C
#10 Alpha® swab with long handle Texwipe® Polyester PP TX761 B
#11 Large Alpha® sampling swab Texwipe® Polyester PP TX714K B
#12 4N6FLOQSwab™ regular flocked swab Copan Nylon flocked Plastic 4520CS01 F (buccal swab)
#13 4N6FLOQSwab™ regular flocked swab Copan Nylon flocked Plastic 3509CS01 F (crime scene)
#14 Sterile foam tipped applicator Puritan® Foam (Polyurethane) PS 25‐1506 1PF F (buccal swab)

DNA Samples

Multiple saliva samples from one donor were isolated using the GeneFiX™ Saliva‐Prep DNA Kit (provided by Isohelix Kent, UK) according to the manufacturers protocol. The concentration of double‐stranded DNA (dsDNA) of the original DNA extract obtained by the GeneFiX™ kit, and after extraction/recovery from the swabs, was determined with the AccuGreen™ High Sensitivity dsDNA Quantitation Kit (provided by Biotium, Fremont, CA, USA, via VWR, Amsterdam, NL). The fluorescence was measured using the Qubit 2.0 fluorometer (Thermo Fischer Scientific, Landsmeer, NL).

Swab Morphology

In order to determine the swab morphology, scanning electron microscopy (SEM) images were taken using a Jeol JSM (6010LA) SEM with an acceleration voltage of 10 kV and with a magnification fixed at 50× (Peabody, MA, USA).

Absorption Capacity

The absorption capacity (AC) was determined by placing the swab directly from the package for 1 min in a tube filled with TE‐buffer (10 mM Tris–HCl; 1 mM EDTA; pH 8.0; Merck, Darmstadt, Germany), where upon the tube was reweighed. With the known density of the TE‐buffer solution ρ (Supplemental Information), the absorption capacity (in μL) of the swabs was determined by:

urn:x-wiley:00221198:media:jfo13837:jfo13837-math-0001(1)

where urn:x-wiley:00221198:media:jfo13837:jfo13837-math-0002 is the difference in average weight (in g) of the tube filled with buffer (urn:x-wiley:00221198:media:jfo13837:jfo13837-math-0003) and the average weight (in g) of the tube with TE‐buffer after removing the swab (urn:x-wiley:00221198:media:jfo13837:jfo13837-math-0004). Each swab was measured five times (n = 5). Details of the protocol are given in the Supplemental Information.

For the extraction and recovery efficiency experiments swabs with an absorption capacity above 100 μL were selected, to ensure that the swab absorbed a sufficient amount of sample and could be premoistened.

Extraction Efficiency

In order to determine the extraction efficiency (EE), 20 μL of DNA from a saliva sample isolated by the GeneFiX™ Saliva‐Prep DNA Kit, was pipetted directly onto each swab. After addition of the dsDNA to the swab, the swab head was cut off and placed in a 1.5 mL Eppendorf tube filled with 250 μL TE‐buffer and then vortexed for about 1 min. A negative control of each swab was taken by replacing the 20 μL of DNA with 20 μL of TE‐buffer. The extraction efficiency (in %) was calculated by:

urn:x-wiley:00221198:media:jfo13837:jfo13837-math-0005(2)

where [sample] is the measured concentration of the DNA sample (in ng/μL), Vapplied the volume (in μL) added to the swab (which is 20 μL, in this case), Vextraction the extraction volume (Supplemental Information) and [origin] the concentration of the original DNA extract (in ng/μL) as obtained with the GeneFix™ kit. Each swab was measured in triplicate (n = 3). More details for extraction and the following recovery protocols can be found in the Supplemental Information.

Recovery Efficiency

In order to determine the recovery efficiency (RE), 20 μL of DNA from a saliva sample isolated with the GeneFiX™ Saliva‐Prep DNA Kit, was pipetted onto a predefined area of a glass slide and dried for about 30 min at room temperature in a sterile laminar flow hood. Swabs were moistened by pipetting 50 μL of TE‐buffer onto the swab head. After the collection step (described in detail in the Supplemental Information), the swab head was cut off and placed in a 1.5 mL Eppendorf tube filled with 250 μL TE‐buffer and vortexed for about 1 min. A negative control was taken for each swab by replacing the 20 μL of DNA with 20 μL of TE‐buffer. RE was determined using an equation very similar to Eq. 2Supplemental Information. Each swab was measured in triplicate (n = 3).

Results and Discussion

Swab Morphology

Scanning electron microscope images of a selection of swabs, to highlight the various design categories as already discussed, are shown in Fig. 1. Images of all tested swabs are presented in Figure S4 of the Supplemental Information. Swab #1 is the only cotton swab used in this research and consists of long wound fibers (Fig. 1A). Three wound rayon swabs, #3, #4, and #5, are tested in this research. The Transwab (#3 Fig. 1B) and the Transport swab (#5) are more densely packed than the Dryswab (#4). The polyester swabs, #7, #9, #10, and #11, show different types of packing (Fig. 1C,D). The Absorbond swab (#7) has a morphology of long wound fibers similar to the cotton swab. The other three swabs have a woven, more pad‐like structure. Swab #2, as well as #12 and #13 (Fig. 1E) are made of nylon‐flocked fibers, which are sprayed onto the swab. The three types of swab have a similar appearance. The nylon Microswab (#6) is a precision swab with a tip diameter of only 0.9 mm consisting of long and thin bundled filaments of nylon. #8 and #14 are foam swabs made of polyurethane and have a very open sponge‐like morphology (Fig. 1F).

image
Scanning electron microscope images of a selection of the tested swab materials. (A) Cotton swab (Eurotubo, #1), (B) Rayon swab (Transwab, #3), (C) Polyester swab (Absorbond, #7), (D) Polyester swab (Honeycomb, #9), (E) Nylon Flocked swab (4N6FLQSwab Crime Scene, #13) and (F) Foam swab (Foam Tipped Applicator, #14).

Absorption Capacity

Most of the swabs show an absorption capacity well above 100 μL, Fig. 2. The Large Alpha polyester swab (#11) has a much larger swabbing area compared to the other swabs, which results in a capacity above 200 μL. Five swabs have a capacity lower than 100 μL, but note that the nylon Microswab (#6) and the polyester Honeycomb swab (#9) have a much smaller swabbing area than the other tested swabs. The rayon Transport swab (#5) and Absorbond polyester swab (#7) do not have an open structure, the fibers are closely packed (see the scanning electron microscope images in Figure S4 in the Supplemental Information). The variation in absorption capacity as found in Fig. 2 does not show a clear relation with swab morphology (see the scanning electron microscope images in Figure S4 in the Supplemental Information). This is due to the fact that the performance of a swab depends not only on swab tip material, but also on the volume and the porosity of the sorbent material, as well as on how tightly the material is wound onto the swab shaft 1.

image
The absorption capacity of the various swabs (n = 5). The error bars are ±1 SD. [Color figure can be viewed at wileyonlinelibrary.com]

Extraction Efficiency

As mentioned before, for the extraction efficiency experiments swabs with an absorption capacity above 100 μL were selected in order to ensure that the swabs absorb sufficient amounts of the sample. The extraction efficiency of these swabs, see Fig. 3 and Table 2, varied greatly with values of between 6% (#13) and 48% (#12), with the nylon flocked 4N6FLOQ swabs showing both the lowest and the highest percentage. It is rather striking that none of the swabs showed a value above 50%, which means that the largest part of the DNA sample remains on the swab. An important point that should be noted here is that in forensic practice it is common to use only one extraction step. Furthermore, it is common to use only a limited extraction volume, to avoid unacceptable high dilution. Inevitably therefore, an extraction efficiency of 100% is impossible, because part of the solution that contains a proportional amount of the dissolved DNA will remain on the swab. A maximum theoretical efficiency was calculated based on the ratio of extraction volume and total volume applied and based on the assumption that the DNA did not adsorb onto the swab surface but was completely dissolved in the buffer solution contained on the swab. In Table S1 of the Supplemental Information the maximum theoretical extraction efficiency is compared to the measured extraction efficiency, from which it is concluded that a substantial part of the DNA stayed behind on the swab. The polyester Alpha swab (#10) and the nylon flocked 4N6FLOQSwab (#12) showed the highest extraction efficiency when the extraction efficiency obtained was compared to the maximum extraction efficiency. Also notable is the low efficiency of swabs #4 and #13, whereas these swabs were specifically designed for forensic purposes.

image
The extraction efficiency of the various swabs (n = 3). The error bars are ±1 SD. [Color figure can be viewed at wileyonlinelibrary.com]
Table 2. Performance of the swabs used in this research. Abs, Absorption @ 260 nm; AC, absorption capacity; C, cotton; CS, crime scene; EE, extraction efficiency; F, foam; N, nylon; NF, nylon flocked; P, polyester; R, rayon; RE, recovery efficiency
Name Type Abs AC (n = 5), μL EE (n = 3), % Concentration (n = 3), ng/μL RE (n = 3), % Concentration (n = 3), ng/μL
#1 EUROTUBO C Yes 121 ± 6 21.5 ± 2.2 1.0 ± 0.2 34.0 ± 11.8 1.1 ± 0.4
#2 eSwab NF Yes 137 ± 7 31.3 ± 10.8 1.3 ± 0.3 40.1 ± 8.1 1.5 ± 0.3
#3 Transwab R Yes 155 ± 12 14.8 ± 7.8 1.1 ± 0.4 18.9 ± 1.0 1.5 ± 0.1
#4 Dryswab R Yes 140 ± 7 15.2 ± 0.3 0.9 ± 0.2 22.5 ± 11.3 1.0 ± 0.4
#5 Transport swab R Yes 84 ± 9 n/a n/a n/a n/a
#6 MicroSwab N No 3 ± 1 n/a n/a n/a n/a
#7 Absorbond P No 60 ± 33 n/a n/a n/a n/a
#8 CleanFoam F No 125 ± 26 34.8 ± 4.9 1.5 ± 0.1 26.9 ± 11.9 1.3 ± 0.2
#9 Honeycomb P No 35 ± 2 n/a n/a n/a n/a
#10 Alpha P No 176 ± 4 34.7 ± 4.8 1.7 ± 0.1 40.0 ± 1.4 1.5 ± 0.2
#11 Large Alpha P No >200 n/a n/a n/a n/a
#12 4N6FLOQ NF Yes 123 ± 9 48.4 ± 2.4 1.8 ± 0.1 46.6 ± 10.0 1.4 ± 0.2
#13 4N6FLOQ CS NF Yes 121 ± 5 5.9 ± 1.9 0.2 ± 0.0 1.5 ± 0.9 0.0 ± 0.0a
#14 Foam tipped F Yes 28 ± 7 n/a n/a n/a n/a
  • a n = 2, due to loss of one swab tip during the experiment.

The measured DNA concentrations for each swab were very similar, with the exception of swab #13, Table 2. Although the swabs were pressed against the tube to obtain as much DNA as possible, the extraction volume for most swabs was less than determined in the absorption capacity experiment, due to the 1 min. of vortexing. Therefore, it was important to take the extraction volume into account in order to determine an accurate extraction efficiency.

A substantial part of the DNA stayed behind on the swabs, resulting in a low extraction efficiency. In literature higher efficiencies were reported for direct inoculation of the swabs with spores. According to Rose et al., using an extraction buffer with a volume of 5 mL, the direct inoculation with Bacillus anthracis spores of cotton, macrofoam and rayon swabs yielded a percentage above 90% and for polyester above 80% 2. Rayon swabs resulted in an extraction efficiency of about 76% with direct inoculation with B. anthracis spores and sonication as the extraction method 10. Adamowicz et al. 17 applied cotton swabs and obtained an extraction efficiency of 50% for a buccal cell suspension and only 20% for a blood sample. Marshall et al. compared the X‐Swab of Diomics with the 4N6FLOQSwab, by applying 1, 2, and 5 ng quantities in a volume of 10 μL on the collection devices. The X‐Swab showed an extraction efficiency of 65%, 48%, and 60% for the aforementioned amounts of DNA, respectively. The 4N6FLOQSwab could recover 85%, 55%, and 32% for these amounts of DNA 19.

Recovery Efficiency

For the recovery efficiency experiments swabs with an absorption capacity above 100 μL were selected, in order to ensure that the swabs absorbed a sufficient amount of sample and could be premoistened. The recovery efficiency of most of these swabs (Fig. 4) was in the range of 20–40%, which meant that the majority of the sample could not be retrieved, a finding confirmed by comparison of the maximum theoretical recovery efficiency (Table S1, Supplemental Information) with the measured recovery efficiency. The nylon‐flocked 4N6FLOQSwab (#12) showed the best recovery efficiency, 47%, the nylon‐flocked 4N6FLOQSwab for crime scene samples (#13) showed the worst performance with an efficiency of only 2%. This was most likely caused by direct binding of DNA to the surface of this swab, in combination with a buffer that was unable to release the bound DNA effectively. However, it should be possible to recover this DNA by a second extraction step in a more suitable buffer. The manufacturer of the nylon flocked 4N6FLOQ Swabs advises the use of a Nucleic Acid Optimizer (NAO) to increase the recovery of DNA from the swab. The NAO is a semi‐permeable basket that is applied in combination with high‐speed centrifugation to collect the eluted DNA sample from the swab 24. Optimization of the recovery efficiency was not the goal of this study; therefore, such a method was not further investigated.

image
The recovery efficiency of the various swabs (n = 3). The error bars are ±1 SD. [Color figure can be viewed at wileyonlinelibrary.com]

Again the polyester Alpha swab (#10) and the nylon flocked 4N6FLOQSwab (#12), together with the nylon flocked eSwab (#2), the rayon Transwab (#3) and the CleanFoam swab (#8), showed the highest extraction efficiency when the extraction efficiency obtained was compared to the maximum extraction efficiency (Table S1, Supplemental Information). As with the extraction efficiency, the recovery efficiency for the measured DNA concentrations did not differ much between the different swab types (Table 2).

DNA recovery efficiencies reported in the literature do vary somewhat, but are usually around 50%. Bacillus anthracis spores were collected from nonporous stainless steel surfaces by Rose et al., resulting in an efficiency above 40% for cotton and macrofoam swabs that were premoistened and vortexed for 2 min 2. For the polyester and rayon swabs an efficiency of around 10% was obtained under the same circumstances 2. Probst et al. 2 also studied Bacillus anthracis spores and obtained 49% recovery with an improved extraction protocol using nylon‐flocked swabs applied to a stainless steel surface. Using the old NASA protocol only about 30% could be recovered, but still higher than with a cotton swab, which only gave a recovery of about 13% 7. The recovery of Bacillus anthracis spores collected with a rayon swab from a stainless steel surface and from a painted wallboard surface resulted both in an efficiency of about 41% according to Brown et al. 10. Using optimal storage conditions Garvin et al. 16 obtained recovery efficiencies of a human saliva sample of 95% and 54% for cotton and viscose swabs, respectively. However, upon isolation the control sample only contained 52% of the predicted amount of DNA. Recovery with cotton and nylon swabs from a petri dish of a saliva sample turned out to be around 60%, an experiment in which a high quantity sample of DNA (more than 500 ng) was deposited 3. Edmonds et al. 8 compared liquid‐deposition and dry aerosol‐deposition of spores on glass. Cotton, dacron, rayon, and foam all gave an efficiency above 60% and 80% for the dry and liquid deposition, respectively. The reported recovery efficiency of the different swap types for bacterial spores and body fluids is in the same range as that for pure DNA (Table 2).

Additional Remarks

A NanoDrop instrument (NanoDrop1000, Thermo Fischer Scientific) can be used to quantify the concentration of DNA in a sample by measuring light absorption of a sample at 260 nm. RNA, ssDNA, and dsDNA all absorb at this wavelength and therefore this method is unable to discriminate between these types of nucleic acids. However, during a NanoDrop measurement it was found that an unknown compound that also absorbs at 260 nm must have leached from the majority of the swabs, Table 2. Therefore, this technique was discarded as a way to determine the extraction and recovery efficiency of swabs.

In order to check if contamination had taken place, negative controls were taken during the extraction and recovery experiments. Indeed some of these blanks gave a substantial concentration value (Table S2, Supplemental Information). A side cutter was used to cut swabs that did not have a suitable breaking point, and tweezers were used to pull out the swab from the tubes. To exclude these cutting tools as a source of DNA contamination, swabs were placed in 200 μL of TE‐buffer for 1 min and then pressed against the side wall to obtain as much extraction volume as possible. The same swabs used in the previous extraction and recovery experiments gave a positive value (Table S2, Supplemental Information) when measured with the Qubit assay. In order to validate if there was any DNA contamination on the swabs a multiple displacement amplification (MDA) reaction was carried out. MDA is an isothermal amplification reaction, which makes use of random hexamer primers that amplify all DNA present in a sample. The applied MDA protocol is described in the Supplemental Information, together with the amplification curve (Figure S5). The DNA concentration of the swab samples was determined prior to amplification with the AccuGreen™ High Sensitivity dsDNA Quantitation Kit and the Qubit assay (Table S2, Supplemental Information). After 1.5 h amplification none of the swab samples showed a positive signal, whilst the positive controls with a similar concentration as the swab samples (based on the Qubit measurements) did show a proper amplification curve. This suggests that the swab material itself caused a false positive reaction with the dye in the AccuGreen™ High Sensitivity dsDNA Quantitation Kit. It is possible that the dye binds to fibers released from the swabs into the extraction volume. The foam swab (#8) did not give a positive value in any of the blank controls.

The performance of several types of swabs may partially be explained by their chemical structure. Cotton and rayon have the same chemical structure (Figure S6, Supplemental Information), but differ in the degree of polymerization with values of 400–700 and 5000 for rayon and cotton, respectively. The polymer in these swab materials contains several O‐H groups, of which it is known that they form hydrogen bonds with nucleic acid chains. Nylon, a polyamide, contains N–H groups that are also known to form hydrogen bonds with nucleic acids. Polyester and polyurethane (foam) have polar C=O groups, which give only weak dipole‐dipole interaction 1425. It is likely though that the absorption capacity of the swabs, even more than on swab material composition, depends on swab tip dimensions and morphology. This is in line with Verdon et al. and Brownlow et al. who conclude that the amount of DNA collected is inversely proportional to the density of the fibers on the swab 13.

Higher DNA recovery efficiencies were obtained by premoistening the swab with e.g., water 131223. The exact efficiency, and therefore the choice of most optimal swab material, is affected by the type of sample (epithelial cells, blood, or saliva), the porosity of the surface, the swab method (use of double swab technique or swab premoistening), swabbing medium (water, isopropanol or other), swabbing angle and the extraction protocol (vortexing or sonication, type of extraction kit) (137101214171823). It should be noted the recovery of more DNA would not necessarily result in good or complete DNA profiles 13.

In addition to DNA loss on the swab itself, determined by the extraction efficiency, some loss of DNA is always to be expected during swabbing, i.e., some DNA on a swabbed surface will not be picked up by the swab. This is most likely the reason that the recovery efficiency was lower for the foam (#8) and two of the nylon flocked swabs (#12 and #13). Note that the number of swabs tested per efficiency experiment was low (n = 5) and the standard deviation relatively high (for some experiments more than 10%). Most of the swabs tested were acquired through donation and therefore only a limited number of swab types was available for testing. In order to achieve a better statistical variance between experiments the efficiencies of two types, the nylon flocked 4N6FLOQSwab (#12) and the foam swab (#8), were tested with a larger data set (n = 10). The extraction and recovery efficiency of the nylon‐flocked 4N6FLOQSwab for this data set were 44.1 ± 7.3% and 40.6 ± 2.1%, respectively. In comparison to the n = 3 test the standard deviation of the extraction efficiency increased, while the recovery efficiency became lower. The n = 10 extraction and recovery efficiencies of the foam swab were 30.6 ± 0.5% and 23.8 ± 6.9%, respectively. Both standard deviations became lower in comparison to the n = 3 test. These standard deviation fluctuations seem to suggest that the variation in extraction and recovery efficiency within one batch of the same swab is relatively high. Nevertheless, it can still be concluded that for many swab types the largest fraction of the DNA sample remained on the swab and could not be retrieved with the chosen protocol.

Alternative options for sample collection that are now available include swabs made of a material that collects high amounts of cells/DNA and subsequently dissolves in the extraction buffer. The recently available X‐Swab from Diomics Corporation dissolves during incubation at 56°C for 1 h. Reported results for blood and saliva samples with this swab show a higher yield and average peak heights after profiling as compared to the 4N6FLOQSwab, whereas there was an indication that the dissolved swab material enhanced the PCR yield 19. Instead of swabs, tape has sometimes been used for sample collection in forensic cases 1113. These minitapes are completely soluble in water, show higher DNA concentrations and full profiles, but a drawback is that the extract is gel‐like, which makes pipetting impossible 13.

Conclusions

Most of the swabs studied showed an absorption capacity well above 100 μL, which is explained by their morphology as determined by SEM. The absorption capacity most likely depends more on the tip dimensions and the winding of the fibers than on the swab material. Despite the high absorption capacity all swabs tested showed an extraction and recovery efficiency for pure DNA of below 50%. The extraction efficiency varied between 5.9% and 48.4% whereas the recovery efficiency was between 1.5% and 46.6%. Overall the nylon flocked 4N6FLOQSwab for buccal samples (#12) performed best with an extraction efficiency of 48% (MTE: 53%), and a recovery efficiency of 47% (MTE: 58%). Upon comparing the obtained efficiencies with the maximum theoretical efficiencies, the nylon flocked 4N6FLOQSwab (#12) and the Polyester Alpha swab (#10) performed equally well. Furthermore it can be concluded that swabs are well designed to allow the pickup of a DNA sample from a surface. However, subsequent extraction of the DNA from the swab is very low compared to the maximum amount of DNA obtainable in theory. This is thought to be due to the chemical interaction of DNA molecules with the swab surface functional groups. In some cases only a limited amount of DNA is present, such as in forensic touch samples. This may result in insufficient DNA becoming available for the determination of a DNA‐profile.

Acknowledgments

The authors thank Dyonne Nijhuis (Saxion University of Applied Sciences) and Celine van de Waardt (Saxion University of Applied Sciences) for their experimental input during their bachelor projects. We would also like to acknowledge the Medical Cell BioPhysics (MCBP) group at the University of Twente for the use of their lab space and their equipment. Many thanks to Mark Smithers (NanoLab cleanroom) for proofreading and English language verification.

source from: https://onlinelibrary.wiley.com/doi/full/10.1111/1556-4029.13837

Leave a Comment

Holotech Medical Supplier

Contact us

Vancouver, BC, Canada

Recent Posts

Our Products

Your Shopping cart

Close