COMMUNICATION
Washed Away; How Not to Lose Your RNA during Isolation Birgitte K. Hønsvall1,2 and Lucy J. Robertson3,* 1
University College of Southeast Norway, Borre, Norway; 2Trilobite Microsystems AS, Borre, Norway; and 3Department of Food Safety and Infection Biology, Norwegian University of Life Sciences, Faculty of Veterinary Medicine, Adamstuen Campus, Oslo, Norway Cryptosporidium oocysts have extremely robust walls that protect the parasite against environmental pressures. Analyses must be sensitive to detect the few organisms (if any) present in environmental samples. After a series of negative nucleic acid amplification results on spiked samples, following a standard RNA isolation protocol, it seemed probable that oocyst RNA had been lost in the washing steps of the isolation protocol. By reducing both the volume of wash buffer and the number of washing steps, positive results could be re-established. Insufficient washing, however, seemed to prevent downstream analysis, probably because of inhibitory substances remaining in the RNA isolate. Nucleic acid isolation protocols for low numbers of “difficult” organisms should be adapted, according to the material to optimize the balance between removal of inhibitors and retention of target, thereby improving the performance of the technique. KEY WORDS: Boom extraction method, Cryptosporidium, Escherichia coli
INTRODUCTION 1
Nucleic acid sequence-based amplification (NASBA) is an isothermal alternative to PCR for amplification of nucleic acids. The target molecule is RNA, rather than DNA, and amplification is carried out at 41°C. Three enzymes are needed for the reaction: avian myeloblastosis virus RT, RNase H, and T7 RNA polymerase. Two primers are also required, and the forward primer has the promoter sequence for T7 RNA polymerase at its 59 end. After the initial RT step, T7 RNA polymerase recognizes the promoter sequence and produces multiple mRNA copies of the template. Exponential amplification occurs by alteration between RNA and DNA molecules. Cryptosporidium oocysts have extremely robust walls that protect the parasite-infective stages—sporozoites— against environmental pressures. The lysing of Cryptosporidium oocysts to obtain nucleic acids is known to require relatively harsh procedures. However, nucleic acids are released upon successful lysis, and analysis of RNA can provide information about both viability and species or strains.2 During RNA analysis of viable Cryptosporidium parvum oocysts using NASBA, replacement of some of the components in the isolation kit resulted in a series of
*ADDRESS CORRESPONDENCE TO: Birgitte K. Hønsvall, Department of Microsystems, University College of Southeast Norway, Raveien 205, 3184 Borre, Norway (E-mail: [email protected]). doi: 10.7171/jbt.17-2802-004
Journal of Biomolecular Techniques 28:75–79 © 2017 ABRF
negative-amplification results being obtained. However, this series of negative results did not start immediately after the replacements. Hence, after ensuring that the lysis and amplification procedures were functioning correctly, the isolation process was investigated. MATERIALS AND METHODS
Oocysts of C. parvum were isolated from feces from infected calves by salt flotation. Escherichia coli K12 was used as a control organism. Cultures were grown in Luria broth medium and harvested after 4 h. Lysis of E. coli was carried out by adding 600 ml NucliSENS lysis buffer (Biom´erieux, Lyon, France) to 50 ml bacterial culture. Lysis of oocysts was carried out by boiling 10–50 ml oocyst suspensions for 1 h in 600 ml NucliSENS lysis buffer, followed by overnight proteinase K treatment at 50°C. The lysed samples of E. coli and C. parvum oocysts were then subject to RNA isolation by the following protocols. The NucliSENS miniMAG (Biom´erieux) isolation kit for total nucleic acids, based on the Boom extraction methodology,3 was the basis for RNA isolation. An overview of the RNA isolation protocol with C. parvum oocysts is shown in Fig. 1. The following replacement components were implemented into the method: c New silica beads from G-Biosciences (Geno Technology, St. Louis, MO, USA) c New Wash Buffer 1 (prepared by mixing 70% ethanol and 30% NucliSENS lysis buffer)
HØNSVALL AND ROBERTSON / WASHED AWAY
FIGURE 1
Overview of the RNA isolation protocol for C. parvum oocysts. Oocysts were first boiled for 1 h in lysis buffer and then treated overnight with proteinase K to break down oocyst walls further and open sporozoite membranes, thereby releasing nucleic acids. Nucleic acids bound to silica beads were washed with wash buffers, according to 3 different protocols (original, Protocol A, and Protocol B; see bottom) before elution.
c New Wash Buffer 2 (prepared by mixing 70% ethanol and 30% RNase-free water) The following is the original isolation protocol, based on the protocol provided by the manufacturer with the kit: to each of the proteinase K-treated oocyst samples or E. coli samples in lysis buffer (610–650 ml in total), 50 ml prepared G-Biosciences silica bead suspension was added and incubated for 10 min. The beads were captured on a magnet and the supernatant discarded. Each sample was then washed twice with 400 ml Wash Buffer 1; twice with 500 ml Wash Buffer 2, and once with 500 ml Buffer 3 (NucliSENS kit). The nucleic acids were eluted into 50 ml elution buffer (NucliSENS kit). Two updated protocols were investigated. The washing steps with Wash Buffers 2 and 3 were modified such that fewer washing steps with less wash buffer were used. Protocol A
To each sample in lysis buffer (610–650 ml in total), 50 ml prepared G-Biosciences silica bead suspension was added and incubated for 10 min. The beads were captured on a magnet and the supernatant discarded. Each sample was 76
then washed twice with 400 ml Wash Buffer 1, once with 400 ml Wash Buffer 2, and once with 400 ml Wash Buffer 3. The nucleic acids were eluted into 50 ml elution buffer (NucliSENS kit). Protocol B
This protocol was identical to Protocol A, except the washing step using Wash Buffer 2 was omitted. (Each sample was washed twice with 400 ml Wash Buffer 1 and once with 400 ml Wash Buffer 3.) To investigate whether RNA had been lost into the eluate and wash buffers, these were not discarded after use but collected and analyzed by NASBA. Real-time NASBA (NucliSENS EasyQ Basic Kit; Biom´erieux) was used for amplification and detection of RNA, with an in-house primer set and molecular beacon targeting the micronemal protein 1 (MIC1) mRNA (primer set “C.par MIC1”).4 Real-time NASBA for E. coli was run with the “Grm neg 16S” primer set.5 Real-time NASBA readings were carried out in a BioTek Synergy 2 reader with Gen5 software (BioTek Instruments, Winooski, VT, USA). The samples were kept JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 2, JULY 2017
HØNSVALL AND ROBERTSON / WASHED AWAY
at 41°C and analyzed every 2 min for 2 h for fluorescent signal from the molecular beacons. The results of NASBA readings were normalized against the negative controls. Samples were interpreted as positive if they fulfilled 3 criteria: 1) they reached a threshold value 1.2 times the negative control6; 2) they had a sigmoidal curve; and 3) they had an increase in relative fluorescence by a factor of 1.7 or more. A sample was regarded as negative if it did not meet all 3 of these criteria. RESULTS AND DISCUSSION
With the use of the original protocol on E. coli (Fig. 2), all eluate and Wash Buffers 2 and 3 gave positive signals. The NASBA result from Wash Buffer 1 was negative, but rapid amplification was noted in Wash Buffer 2. This amplification was even faster than that in the eluted sample, although not reaching the same level of relative fluorescence units (RFU). The running of NASBA on collected Wash Buffer 1 gave negative results for both E. coli and C. parvum. The lysis buffer and Wash Buffer 1 contain the chaotropic agent guanidinium thiocyanate (GuSCN), which assists in the binding of the nucleic acids binding to silica particles and inhibits RNases.3, 7 Thus, Wash Buffer 1 would be less likely to result in loss of nucleic acids. As the concentration of the chaotropic agent is reduced throughout the multiple washing steps, nucleic acids will bind less strongly to the silica beads. Whereas the replaced Wash Buffer 2 consisted of 70% ethanol,3 Wash Buffer 2 in the kit is 2-(Nmorpholino)ethanesulfonic acid buffer (Biom´erieux). This buffer has a mid-range pKa, minimal salt effects, and binds only weakly to Ca, Mg, and Mn; it might wash out less RNA than 70% ethanol. Sun and coworkers7 reported a
decrease in RNA yield in their isolation protocol after washing with 70% ethanol. They also experienced that the initial binding of nucleic acids to silica beads did not occur in 70% ethanol without the addition of GuSCN. Although GuSCN is crucial for binding of nucleic acids to silica and for preventing digestion of the RNA by RNases, GuSCN might also hamper the enzymes used in the amplification protocol. Wash Buffer 3 contains borate buffer, and in this wash step, the nucleic acids are prepared for elution. The positive NASBA results for Buffer 3 are therefore not unexpected. As Wash Buffer 2 and 3 gave positive NASBA results, it is reasonable to assume that these 2 buffers do not contain any inhibitors that may affect the downstream amplification reaction. Therefore, the NASBA amplification results seem to depend on 2 factors: 1) minimal loss of RNA in the washing steps and 2) sufficient removal of inhibitors (from the sample itself and from lysis buffer and Wash Buffer 1). RNA yield is maximized when the balance between these 2 contrasting factors is optimal. It would be interesting to investigate the effect of adding wash buffers to known amounts of RNA. However, we did not have the opportunity to investigate this. With the use of the original protocol, none of the C. parvum samples investigated (from 105 to 955 oocysts) gave a positive result by NASBA. However, when the washing steps were reduced (Protocol A), all samples (down to 140 oocysts per sample; this was the lowest concentration tested) gave a positive signal (Fig. 3A). However, positive signals were also obtained in Wash Buffer 3. In the control with E. coli, positive NASBA results were obtained from both Wash Buffers 2 and 3.
FIGURE 2
Real-time NASBA amplification of 16S rRNA of E. coli using the original isolation protocol. The data are normalized against the negative control.
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HØNSVALL AND ROBERTSON / WASHED AWAY
FIGURE 3
Real-time NASBA amplification of MIC1 mRNA from C. parvum oocysts using Protocol A (A) and Protocol B (B) for RNA isolation. Eluted RNA contains RNA from maximum 5000 oocysts, and the diluted eluted RNA 1:5 contains RNA from maximum 1000 oocysts.
Although these results suggest washing steps should be minimized, further reduction in the washing steps to minimize loss of target might result in insufficient removal of inhibitory substances. Negative NASBA results were often obtained from samples containing E. coli and C. parvum oocysts when Protocol B was used for RNA isolation (Fig. 3B). However, 1:5 dilution of these eluted samples produced positive results, indicating that inhibitors were present. In contrast, when Protocol A was used for washing, the same samples were positive, both undiluted and diluted. NASBA has been reported to be less sensitive to inhibition than PCR,8, 9 but inhibitory substances can still impede the amplification. The use of the Boom extraction method to isolate RNA has also been reported to abolish inhibition of RT-PCR because of the extensive washing procedure.8 78
The problems that we experienced with RNA isolation and overcame by altering the washing steps are unlikely to be exclusive to Cryptosporidium. This challenge is also likely to be relevant for other organisms that require harsh lysis methods and may result in small quantities of nucleic acids being released; this could include, but is not limited to, other pathogens that spread in the environment as cysts, oocysts, or spores. These organisms are often dispersed in the environment, and the low number of organisms available for analysis compared with the quantity of potential inhibitors in the sample material might further affect the sensitivity of any detection method. Thus, optimization of washing protocols to obtain the best balance between retention of target nucleic acids and removal of inhibitors is essential. In conclusion, the loss of RNA was decreased by using a modified wash procedure during nucleic acid isolation. The JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 2, JULY 2017
HØNSVALL AND ROBERTSON / WASHED AWAY
modifications were essentially reducing the volumes of wash buffer used and using fewer washes. This enabled successful amplification to be achieved. However, too few washes may result in insufficient removal of inhibitory substances, and these may impede downstream analysis. ACKNOWLEDGMENTS This work was funded by the industrial Ph.D. program of The Research Council of Norway (Grant Number 225680/o30) and Trilobite Microsystems AS.
DISCLOSURES
4.
5.
6.
7.
The authors declare that there are no conflicts of interest. REFERENCES 1. Compton J. Nucleic acid sequence-based amplification. Nature 1991;350:91–92. 2. Baeumner AJ, Humiston MC, Montagna RA, Durst RA. Detection of viable oocysts of Cryptosporidium parvum following nucleic acid sequence based amplification. Anal Chem 2001;73:1176–1180. 3. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 2, JULY 2017
8.
9.
purification of nucleic acids. J Clin Microbiol 1990;28: 495–503. Hønsvall BK, Robertson LJ. Real-time nucleic acid sequencebased amplification (NASBA) assay targeting MIC1 for detection of Cryptosporidium parvum and Cryptosporidium hominis oocysts. Exp Parasitol 2017;172:61–67. Zhao Y, Park S, Kreiswirth BN, Ginocchio CC, Veyret R, Laayoun A, Troesch A, Perlin DS. Rapid real-time nucleic acid sequence-based amplification-molecular beacon platform to detect fungal and bacterial bloodstream infections. J Clin Microbiol 2009;47:2067–2078. Landry ML, Garner R, Ferguson D. Real-time nucleic acid sequence-based amplification using molecular beacons for detection of enterovirus RNA in clinical specimens. J Clin Microbiol 2005;43:3136–3139. Sun N, Deng C, Liu Y, Zhao X, Tang Y, Liu R, Xia Q, Yan W, Ge G. Optimization of influencing factors of nucleic acid adsorption onto silica-coated magnetic particles: application to viral nucleic acid extraction from serum. J Chromatogr A 2014; 1325:31–39. Dyer JR, Gilliam BL, Eron JJ Jr, Grosso L, Cohen MS, Fiscus SA. Quantitation of human immunodeficiency virus type 1 RNA in cell free seminal plasma: comparison of NASBA with Amplicor reverse transcription-PCR amplification and correlation with quantitative culture. J Virol Methods 1996;60:161–170. Rutjes SA, van den Berg HH, Lodder WJ, de Roda Husman AM. Real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay. Appl Environ Microbiol 2006;72:5349–5358.
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Washed Away; How Not to Lose Your RNA during Isolation Birgitte K. Hønsvall1,2 and Lucy J. Robertson3,* 1
University College of Southeast Norway, Borre, Norway; 2Trilobite Microsystems AS, Borre, Norway; and 3Department of Food Safety and Infection Biology, Norwegian University of Life Sciences, Faculty of Veterinary Medicine, Adamstuen Campus, Oslo, Norway Cryptosporidium oocysts have extremely robust walls that protect the parasite against environmental pressures. Analyses must be sensitive to detect the few organisms (if any) present in environmental samples. After a series of negative nucleic acid amplification results on spiked samples, following a standard RNA isolation protocol, it seemed probable that oocyst RNA had been lost in the washing steps of the isolation protocol. By reducing both the volume of wash buffer and the number of washing steps, positive results could be re-established. Insufficient washing, however, seemed to prevent downstream analysis, probably because of inhibitory substances remaining in the RNA isolate. Nucleic acid isolation protocols for low numbers of “difficult” organisms should be adapted, according to the material to optimize the balance between removal of inhibitors and retention of target, thereby improving the performance of the technique. KEY WORDS: Boom extraction method, Cryptosporidium, Escherichia coli
INTRODUCTION 1
Nucleic acid sequence-based amplification (NASBA) is an isothermal alternative to PCR for amplification of nucleic acids. The target molecule is RNA, rather than DNA, and amplification is carried out at 41°C. Three enzymes are needed for the reaction: avian myeloblastosis virus RT, RNase H, and T7 RNA polymerase. Two primers are also required, and the forward primer has the promoter sequence for T7 RNA polymerase at its 59 end. After the initial RT step, T7 RNA polymerase recognizes the promoter sequence and produces multiple mRNA copies of the template. Exponential amplification occurs by alteration between RNA and DNA molecules. Cryptosporidium oocysts have extremely robust walls that protect the parasite-infective stages—sporozoites— against environmental pressures. The lysing of Cryptosporidium oocysts to obtain nucleic acids is known to require relatively harsh procedures. However, nucleic acids are released upon successful lysis, and analysis of RNA can provide information about both viability and species or strains.2 During RNA analysis of viable Cryptosporidium parvum oocysts using NASBA, replacement of some of the components in the isolation kit resulted in a series of
*ADDRESS CORRESPONDENCE TO: Birgitte K. Hønsvall, Department of Microsystems, University College of Southeast Norway, Raveien 205, 3184 Borre, Norway (E-mail: [email protected]). doi: 10.7171/jbt.17-2802-004
Journal of Biomolecular Techniques 28:75–79 © 2017 ABRF
negative-amplification results being obtained. However, this series of negative results did not start immediately after the replacements. Hence, after ensuring that the lysis and amplification procedures were functioning correctly, the isolation process was investigated. MATERIALS AND METHODS
Oocysts of C. parvum were isolated from feces from infected calves by salt flotation. Escherichia coli K12 was used as a control organism. Cultures were grown in Luria broth medium and harvested after 4 h. Lysis of E. coli was carried out by adding 600 ml NucliSENS lysis buffer (Biom´erieux, Lyon, France) to 50 ml bacterial culture. Lysis of oocysts was carried out by boiling 10–50 ml oocyst suspensions for 1 h in 600 ml NucliSENS lysis buffer, followed by overnight proteinase K treatment at 50°C. The lysed samples of E. coli and C. parvum oocysts were then subject to RNA isolation by the following protocols. The NucliSENS miniMAG (Biom´erieux) isolation kit for total nucleic acids, based on the Boom extraction methodology,3 was the basis for RNA isolation. An overview of the RNA isolation protocol with C. parvum oocysts is shown in Fig. 1. The following replacement components were implemented into the method: c New silica beads from G-Biosciences (Geno Technology, St. Louis, MO, USA) c New Wash Buffer 1 (prepared by mixing 70% ethanol and 30% NucliSENS lysis buffer)
HØNSVALL AND ROBERTSON / WASHED AWAY
FIGURE 1
Overview of the RNA isolation protocol for C. parvum oocysts. Oocysts were first boiled for 1 h in lysis buffer and then treated overnight with proteinase K to break down oocyst walls further and open sporozoite membranes, thereby releasing nucleic acids. Nucleic acids bound to silica beads were washed with wash buffers, according to 3 different protocols (original, Protocol A, and Protocol B; see bottom) before elution.
c New Wash Buffer 2 (prepared by mixing 70% ethanol and 30% RNase-free water) The following is the original isolation protocol, based on the protocol provided by the manufacturer with the kit: to each of the proteinase K-treated oocyst samples or E. coli samples in lysis buffer (610–650 ml in total), 50 ml prepared G-Biosciences silica bead suspension was added and incubated for 10 min. The beads were captured on a magnet and the supernatant discarded. Each sample was then washed twice with 400 ml Wash Buffer 1; twice with 500 ml Wash Buffer 2, and once with 500 ml Buffer 3 (NucliSENS kit). The nucleic acids were eluted into 50 ml elution buffer (NucliSENS kit). Two updated protocols were investigated. The washing steps with Wash Buffers 2 and 3 were modified such that fewer washing steps with less wash buffer were used. Protocol A
To each sample in lysis buffer (610–650 ml in total), 50 ml prepared G-Biosciences silica bead suspension was added and incubated for 10 min. The beads were captured on a magnet and the supernatant discarded. Each sample was 76
then washed twice with 400 ml Wash Buffer 1, once with 400 ml Wash Buffer 2, and once with 400 ml Wash Buffer 3. The nucleic acids were eluted into 50 ml elution buffer (NucliSENS kit). Protocol B
This protocol was identical to Protocol A, except the washing step using Wash Buffer 2 was omitted. (Each sample was washed twice with 400 ml Wash Buffer 1 and once with 400 ml Wash Buffer 3.) To investigate whether RNA had been lost into the eluate and wash buffers, these were not discarded after use but collected and analyzed by NASBA. Real-time NASBA (NucliSENS EasyQ Basic Kit; Biom´erieux) was used for amplification and detection of RNA, with an in-house primer set and molecular beacon targeting the micronemal protein 1 (MIC1) mRNA (primer set “C.par MIC1”).4 Real-time NASBA for E. coli was run with the “Grm neg 16S” primer set.5 Real-time NASBA readings were carried out in a BioTek Synergy 2 reader with Gen5 software (BioTek Instruments, Winooski, VT, USA). The samples were kept JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 2, JULY 2017
HØNSVALL AND ROBERTSON / WASHED AWAY
at 41°C and analyzed every 2 min for 2 h for fluorescent signal from the molecular beacons. The results of NASBA readings were normalized against the negative controls. Samples were interpreted as positive if they fulfilled 3 criteria: 1) they reached a threshold value 1.2 times the negative control6; 2) they had a sigmoidal curve; and 3) they had an increase in relative fluorescence by a factor of 1.7 or more. A sample was regarded as negative if it did not meet all 3 of these criteria. RESULTS AND DISCUSSION
With the use of the original protocol on E. coli (Fig. 2), all eluate and Wash Buffers 2 and 3 gave positive signals. The NASBA result from Wash Buffer 1 was negative, but rapid amplification was noted in Wash Buffer 2. This amplification was even faster than that in the eluted sample, although not reaching the same level of relative fluorescence units (RFU). The running of NASBA on collected Wash Buffer 1 gave negative results for both E. coli and C. parvum. The lysis buffer and Wash Buffer 1 contain the chaotropic agent guanidinium thiocyanate (GuSCN), which assists in the binding of the nucleic acids binding to silica particles and inhibits RNases.3, 7 Thus, Wash Buffer 1 would be less likely to result in loss of nucleic acids. As the concentration of the chaotropic agent is reduced throughout the multiple washing steps, nucleic acids will bind less strongly to the silica beads. Whereas the replaced Wash Buffer 2 consisted of 70% ethanol,3 Wash Buffer 2 in the kit is 2-(Nmorpholino)ethanesulfonic acid buffer (Biom´erieux). This buffer has a mid-range pKa, minimal salt effects, and binds only weakly to Ca, Mg, and Mn; it might wash out less RNA than 70% ethanol. Sun and coworkers7 reported a
decrease in RNA yield in their isolation protocol after washing with 70% ethanol. They also experienced that the initial binding of nucleic acids to silica beads did not occur in 70% ethanol without the addition of GuSCN. Although GuSCN is crucial for binding of nucleic acids to silica and for preventing digestion of the RNA by RNases, GuSCN might also hamper the enzymes used in the amplification protocol. Wash Buffer 3 contains borate buffer, and in this wash step, the nucleic acids are prepared for elution. The positive NASBA results for Buffer 3 are therefore not unexpected. As Wash Buffer 2 and 3 gave positive NASBA results, it is reasonable to assume that these 2 buffers do not contain any inhibitors that may affect the downstream amplification reaction. Therefore, the NASBA amplification results seem to depend on 2 factors: 1) minimal loss of RNA in the washing steps and 2) sufficient removal of inhibitors (from the sample itself and from lysis buffer and Wash Buffer 1). RNA yield is maximized when the balance between these 2 contrasting factors is optimal. It would be interesting to investigate the effect of adding wash buffers to known amounts of RNA. However, we did not have the opportunity to investigate this. With the use of the original protocol, none of the C. parvum samples investigated (from 105 to 955 oocysts) gave a positive result by NASBA. However, when the washing steps were reduced (Protocol A), all samples (down to 140 oocysts per sample; this was the lowest concentration tested) gave a positive signal (Fig. 3A). However, positive signals were also obtained in Wash Buffer 3. In the control with E. coli, positive NASBA results were obtained from both Wash Buffers 2 and 3.
FIGURE 2
Real-time NASBA amplification of 16S rRNA of E. coli using the original isolation protocol. The data are normalized against the negative control.
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HØNSVALL AND ROBERTSON / WASHED AWAY
FIGURE 3
Real-time NASBA amplification of MIC1 mRNA from C. parvum oocysts using Protocol A (A) and Protocol B (B) for RNA isolation. Eluted RNA contains RNA from maximum 5000 oocysts, and the diluted eluted RNA 1:5 contains RNA from maximum 1000 oocysts.
Although these results suggest washing steps should be minimized, further reduction in the washing steps to minimize loss of target might result in insufficient removal of inhibitory substances. Negative NASBA results were often obtained from samples containing E. coli and C. parvum oocysts when Protocol B was used for RNA isolation (Fig. 3B). However, 1:5 dilution of these eluted samples produced positive results, indicating that inhibitors were present. In contrast, when Protocol A was used for washing, the same samples were positive, both undiluted and diluted. NASBA has been reported to be less sensitive to inhibition than PCR,8, 9 but inhibitory substances can still impede the amplification. The use of the Boom extraction method to isolate RNA has also been reported to abolish inhibition of RT-PCR because of the extensive washing procedure.8 78
The problems that we experienced with RNA isolation and overcame by altering the washing steps are unlikely to be exclusive to Cryptosporidium. This challenge is also likely to be relevant for other organisms that require harsh lysis methods and may result in small quantities of nucleic acids being released; this could include, but is not limited to, other pathogens that spread in the environment as cysts, oocysts, or spores. These organisms are often dispersed in the environment, and the low number of organisms available for analysis compared with the quantity of potential inhibitors in the sample material might further affect the sensitivity of any detection method. Thus, optimization of washing protocols to obtain the best balance between retention of target nucleic acids and removal of inhibitors is essential. In conclusion, the loss of RNA was decreased by using a modified wash procedure during nucleic acid isolation. The JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 2, JULY 2017
HØNSVALL AND ROBERTSON / WASHED AWAY
modifications were essentially reducing the volumes of wash buffer used and using fewer washes. This enabled successful amplification to be achieved. However, too few washes may result in insufficient removal of inhibitory substances, and these may impede downstream analysis. ACKNOWLEDGMENTS This work was funded by the industrial Ph.D. program of The Research Council of Norway (Grant Number 225680/o30) and Trilobite Microsystems AS.
DISCLOSURES
4.
5.
6.
7.
The authors declare that there are no conflicts of interest. REFERENCES 1. Compton J. Nucleic acid sequence-based amplification. Nature 1991;350:91–92. 2. Baeumner AJ, Humiston MC, Montagna RA, Durst RA. Detection of viable oocysts of Cryptosporidium parvum following nucleic acid sequence based amplification. Anal Chem 2001;73:1176–1180. 3. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 28, ISSUE 2, JULY 2017
8.
9.
purification of nucleic acids. J Clin Microbiol 1990;28: 495–503. Hønsvall BK, Robertson LJ. Real-time nucleic acid sequencebased amplification (NASBA) assay targeting MIC1 for detection of Cryptosporidium parvum and Cryptosporidium hominis oocysts. Exp Parasitol 2017;172:61–67. Zhao Y, Park S, Kreiswirth BN, Ginocchio CC, Veyret R, Laayoun A, Troesch A, Perlin DS. Rapid real-time nucleic acid sequence-based amplification-molecular beacon platform to detect fungal and bacterial bloodstream infections. J Clin Microbiol 2009;47:2067–2078. Landry ML, Garner R, Ferguson D. Real-time nucleic acid sequence-based amplification using molecular beacons for detection of enterovirus RNA in clinical specimens. J Clin Microbiol 2005;43:3136–3139. Sun N, Deng C, Liu Y, Zhao X, Tang Y, Liu R, Xia Q, Yan W, Ge G. Optimization of influencing factors of nucleic acid adsorption onto silica-coated magnetic particles: application to viral nucleic acid extraction from serum. J Chromatogr A 2014; 1325:31–39. Dyer JR, Gilliam BL, Eron JJ Jr, Grosso L, Cohen MS, Fiscus SA. Quantitation of human immunodeficiency virus type 1 RNA in cell free seminal plasma: comparison of NASBA with Amplicor reverse transcription-PCR amplification and correlation with quantitative culture. J Virol Methods 1996;60:161–170. Rutjes SA, van den Berg HH, Lodder WJ, de Roda Husman AM. Real-time detection of noroviruses in surface water by use of a broadly reactive nucleic acid sequence-based amplification assay. Appl Environ Microbiol 2006;72:5349–5358.
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