- Email: [email protected]
Rapid DNA extraction for molecular epidemiological studies of malaria
Acta Tropica 72 (1999) 149 – 155
Rapid DNA extraction for molecular epidemiological studies of malaria Lars Henning a,b, Ingrid Felger a, Hans-Peter Beck a,* b
a Swiss Tropical Institute, Socinstrasse 57, 4002 Basel, Switzerland Uni6ersity of Witten-Herdecke, Stockumerstrasse 10, D-58448 Witten, Germany
Received 20 May 1998; received in revised form 8 October 1998; accepted 19 October 1998
Abstract DNA isolation from blood samples collected in molecular epidemiological studies is crucial for the quality and reproducibility of data. Blood samples from two malaria endemic sites have been prepared by four different DNA isolation methods with subsequent PCR amplification of the msp2 locus of Plasmodium falciparum. We tested a rapid boiling method; the guanadine isothiocyanate DNA extraction; QIAmp™ blood kit; and the ISOCODE™ STIX PCR template preparation dipstick, and analysed the numbers of concurrent infections/sample. The rapid boiling method and the ISOCODE™ STIX provided overall the best sensitivity combined with ease of handling. The possibility to store and ship the ISOCODE™ STIX at ambient temperature adds further advantage to this method. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: DNA extraction; Molecular epidemiology; Malaria; Polymerase chain reaction; msp2 ; Genotyping; Plasmodium falciparum
Abbre6iations: GTC, guanidine isothiocyanate; MSP 2, merozoite surface protein 2; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism. * Corresponding author. Tel.: + 41-61-284-8116; fax: + 41-61-271-8654; e-mail: [email protected]. 0001-706X/99/$ - see front matter © 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 9 8 ) 0 0 0 9 0 - 4
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1. Introduction Over the past few years PCR-based genotyping of pathogens has become a central technique applied in diagnostics and in molecular epidemiological studies. PCR amplification of a marker sequence is a convenient and practical tool in epidemiological studies, where only limited amounts of specimen, such as blood, are available. The extraction of DNA from such samples is a critical step and several methods are being used. In malaria field studies involving detection of parasite DNA and genotyping of the infecting parasites, mostly finger prick blood samples are collected (Felger et al., 1994; Beck et al., 1997). DNA is routinely isolated from these samples either by phenol/chloroform extraction or by a rapid boiling method (Foley et al., 1992). Methods for DNA preparations from blood samples in studies of epidemiological scale have to fulfil the following criteria: (1) rapid preparation and large through put, (2) high reliability, (3) production of DNA of good quality for long-term storage, (4) avoidance of cross-contamination, (5) reasonable costs. In this study we compared four different methods of DNA preparation from samples collected during malaria field studies in order to test for their reliability, sensitivity, and ease of handling. The following methods were compared: rapid boiling method; guanidine isothiocyanate (GTC) preparation with subsequent phenol/chloroform extraction; a commercially available DNA purification kit (QIAamp® Blood Kit Cat.No.29104, Qiagen, Basle, Switzerland); and a new dipstick for DNA preparation (ISOCODE™ STIX PCR Template Preparation Dipstick, Schleicher&Schuell). The ISOCODE™ dipstick also allows storage and transportation at ambient temperature and thus was expected to be a practical tool for field studies. Because of this advantage for field studies we compared DNA obtained from the ISOCODE™ STIX for PCR amplification with DNA prepared by the other three standard extraction methods in order to assess reliability and sensitivity. DNAs prepared by these different techniques were compared in a standard PCR amplification of the merozoite surface protein (MSP2) locus of Plasmodium falciparum. msp2 has been frequently used in genotyping studies as highly polymorphic marker (Felger et al., 1994; Contamin et al., 1996). In blood samples from areas of high malaria endemicity, multiple P. falciparum infections have been frequently observed (Felger et al., 1994). As a measurement for sensitivity we used the number of msp2 genotypes detected after PCR amplification of templates prepared by the different extraction methods. Because of the lack of fresh infected whole blood, DNA was extracted from 40 pellets of packed cells which had been stored at −20°C. Red blood cells were diluted with serum-free RPMI in order to reconstitute the red blood cell concentration. Packed cells were derived from blood samples collected in Papua New Guinea (10) and Tanzania (30) during two field studies. Parasitaemia levels within the 40 samples ranged from 1 to 739 parasites/200 leucocytes. Nine samples were microscopy negative.
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2. Template preparation
2.1. QIAamp ® Blood Kit (Cat.No.29104, Qiagen, Basle, Switzerland) The kit was used according to the supplier’s instruction. Briefly, 10 ml of blood were diluted in 190 ml phosphate-buffered saline (PBS). Two hundred microlitres of Buffer AL and 25 ml of the provided Proteinase K were added and immediately vortexed for 15 s. After incubating for 10 min at 70°C, DNA was precipitated with 210 ml 96% ethanol. The mixture was applied to a QIAamp® spin column and after centrifugation, the column was washed twice with Buffer AW. DNA was eluted with 50 ml dH2O at 70°C. Five microlitres of this DNA solution were used as template for PCR amplification. Affinity purification yields good quality DNA, but is comparatively expensive.
2.2. Rapid boiling method Ten microlitres of blood were added to 500 ml ice-cold 5 mM sodium phosphate buffer (pH 8.0), vortexed and spun for 2 min at 14 000 × g. The supernatant was discarded and washing was repeated twice. Fifty microlitres dH2O were added to the washed pellet, and after resuspension, boiled for 10 min. Finally, the sample was spun for 10 min at 14 000×g and the pellet discarded. Five microlitres of the supernatant were used for PCR amplification. This method is very fast and inexpensive, nevertheless long-term storage affects the quality of the DNA isolated by this method.
2.3. GTC/phenol/chloroform extraction Fifty microlitres GTC (4 M guanidine isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sodium sarcosylsufate) and 5 ml of 2 M sodium acetate (pH 4.4) were added to 10 ml of blood. Subsequently, 50 ml phenol (pH 8, equilibrated with Ph-Maker™, Oncor-Appligene, France) and 50 ml chloroform were added, the mixture was vortexed and placed on ice for 15 min. The sample was spun for 10 min at 14 000 ×g, the aqueous phase transferred to a new tube and precipitated with 100 ml isopropanol. After centrifugation for 20 min at 14 000× g, the supernatant was discarded and the pellet washed with 200 ml 70% ethanol and spun again. The air-dried pellet was dissolved in 50 ml dH2O and 5 ml were used for PCR amplification. This method is time consuming and prone to contamination due to frequent pipetting steps and changes of tubes.
2.4. ISOCODE™ STIX PCR Template Preparation Dipstick (Cat.No. 495020, Schleicher & Schuell, Dassel, Germany) DNA was extracted from the dipstick according to the supplier’s instructions. Briefly, 10 ml of blood were loaded onto one triangle of the dipstick and dried for 3 h at room temperature. The triangle was clipped off and transferred into a
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L. Henning et al. / Acta Tropica 72 (1999) 149–155
microfuge tube and washed twice with 500 ml dH2O by vortexing three times for at least 5 s. Vortexing less than 5 s may lead to DNA of lower quality impeding subsequent amplification. After complete removal of dH2O, the tube was spun for 5 s and residual water was pipetted off. Fifty microlitres of dH2O were added and incubated for 30 min at 95°C. Finally, the tube was gently tapped 20 times and the supernatant transferred into a new tube. PCR amplification was performed with 5 ml DNA solution. We also added the complete triangle after two washes into the PCR-reaction with similar results or with increased sensitivity.
2.5. PCR amplification PCR amplification of the msp2 locus was performed as described previously (Felger et al., 1994). In all experiments, 5 ml DNA template were used in the primary PCR reaction and 2 ml primary product were used in a 100 ml nested PCR reaction using a Perkin Elmer DNA Thermal Cycler 480. Ten microlitres of nested product were loaded onto a 10% polyacrylamide gel, run, stained with ethidium bromide, and visualized by UV-illumination.
2.6. Genotyping Fifteen microlitres nested PCR product were digested with Hinf I (Biolabs, New England), producing a genotype-specific RFLP-pattern (Felger et al., 1999). For higher resolution and discrimination power, e.g. in the case of a high number of concurrent infections, additional Dde I-, Rsa I-, and ScrF I-digests (all Biolabs) were performed. Restriction fragments were separated on a 10% polyacrylamide gel. For determination of fragment lengths a 1 kb DNA ladder (Gibco-Lifetechnologies, Basle, Switzerland) was used. For quality control, primary and nested PCR from 10% of the samples was repeated and negative samples were all repeated once with equal results. The analysis was carried out by two persons independently. A full comparison of 40 blood samples, including samples negative and positive for P. falciparum by microscopy, was performed using DNA purified with QIAamp® blood kit and ISOCODE™ STIX. The numbers of concurrent infections (multiplicity) defined by msp2 genotyping in these 40 samples are listed in Table 1. The average number of genotypes found per sample with ISOCODE™ STIX preparation was higher than with the QIAamp® preparation. However, this difference was not statistically significant. Nineteen samples had equal multiplicity when the amplification results of the two extraction methods were compared. With the applied RFLP typing scheme, msp2 alleles can be classified into two distinct allelic families: FC27 and 3D7. There was no difference in the detection sensitivity for both families with both template preparations. Fig. 1 depicts the correlation between numbers of alleles found by either method. There were five samples found negative with QIAamp® prepared template, and which were positive using ISOCODE™ STIX prepared template. Two of these were negative by microscopy, the remaining three had low parasite densities (53 parasites/200 leucocytes).
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153
A subset of 12 samples were further used to compare template preparation with GTC/phenol/chloroform extraction and with the rapid boiling method. Table 2 shows the mean number of msp2 alleles/sample detected using templates prepared with all four DNA preparations. Differences found were not statistically significant. Furthermore, the number of alleles detected in each sample was used to establish a ranking for the sensitivity of the four methods. Six out of 12 samples tested had an equal number detected by all four methods. The rapid boiling method and the ISOCODE™ STIX provided the best result in four samples. Within the remaining two samples either QIAamp® or GTC /phenol/chloroform extraction gave the best results on their own. Hence, using the rapid boiling method or the ISOCODE™ STIX method provides the most sensitive determination of overall multiplicity. In order to determine the detection limit of the ISOCODE™ STIX preparation method, serial dilutions of packed cells from blood with a parasitaemia of 5% were made in PBS starting from 10 ml of packed cells. The detection limit for msp2 PCR amplification was calculated on the assumption that 1% parasitaemia corresponds to 50 000 parasites/ml blood. The greatest dilution of the template which still produced an amplification product using 10 ml of template was 1:100 000, which corresponds to 2.5 parasites/ml blood (parasitaemia of 0.00005%). This is in a comparable range of sensitivity reported by others (Foley et al., 1992; Snounou et al., 1993). However, the detection limit by PCR varies depending on the specific gene chosen for amplification. Binding capacity of the ISOCODE™ STIX was also examined. Therefore, the sample application area of the dipstick was loaded with 4, 6, 8, 10, 12, 15, 20, 25 ml whole blood from a single blood sample, respectively. The amount of loaded blood did not affect the detection, and one genotype was detected throughout all samples. Furthermore, storage of template prepared using the ISOCODE™ STIX for 2 months at + 4°C did not affect the template quality and equal results were obtained when tested on four positive samples. Our study showed that template preparation by the rapid boiling method or using the ISOCODE™ STIX provided increased sensitivity for the detection of multiple infections when compared to QIAamp® or GTC/phenol/chloroform template preparation. Although the rapid boiling method offers high sensitivity and Table 1 Comparison of two different DNA extraction methods with respect to the number of msp2 alleles detected in 40 samplesa
Mean multiplicity ( 9 SE) Mean FC27 multiplicity ( 9 SE) Mean 3D7 multiplicity ( 9 SE) Positive samples Higher multiplicity found by the respective method a
QIAamp®
ISOCODE™ STIX
2.38 9 0.33* 1.23 90.18* 1.15 9 0.17* 28 8/21
2.58 9 0.33* 1.2390.17* 1.28 9 0.20* 33 13/21
Multiplicity, number of genotypes detected per blood sample; SE, standard error. * Difference not significant.
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Fig. 1. Comparison of number of alleles found by ISOCODE™ STIX and QIAamp® in 40 samples.
cost-effectiveness, long-term storage of template is critical. The ISOCODE™ STIX offers good sensitivity and in addition, convenient sample handling and circumvents the need for transportation of blood samples using a cold chain. We have carried out studies in Papua New Guinea, Sao Tome and Tanzania successfully using the ISOCODE™ STIX, which was tested by also using frozen EDTA-blood. The DNA preparation is fast, taking less than 1 h, and is easily performed. The prepared DNA was shown to be of high quality and allows long-term storage. Furthermore, minimal handling steps minimize the risk of cross-contamination. The good performance and the additional advantages of the ISOCODE™ STIX seem to make it a reliable tool for molecular epidemiological studies involving PCR and would offer a convenient method to standardize such genotyping studies, which is urgently needed.
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Table 2 Mean multiplicity of infections detected by four different DNA extraction methods Extraction method
Sample size
Total alleles
Mean multiplicity*
Rapid boiling method GTC/Phenol/Chloroform QIAamp® ISOCODE™ STIX
12 12 12 12
26 21 19 24
2.17 90.57 1.75 90.53 1.58 90.52 2.00 90.59
* Differences between the methods were not statistically significant.
Acknowledgements Lars Henning was supported by the Daniela und Ju¨rgen Westphal-Stiftung. This work obtained financial support from the German Science Foundation (DFG) grant no. BE1075/2-1.
References Beck, H.P., Felger, I., Huber, W., Steiger, S., Smith, T., Weiss, N., Alonso, P., Tanner, M., 1997. Analysis of multiple Plasmodium falciparum infections in Tanzanian children during the phase III trail of the malaria vaccine SPf66. J. Infect. Dis. 175, 921 – 926. Contamin, H., Fandeur, T., Rogier, C., Bonnefoy, S., Konate, L., Trape, J.F., Mercereau-Puijalon, O., 1996. Different genetic characteristics of Plasmodium falciparum isolates collected during successive clinical malaria episodes in Senegales children. Am. J. Trop. Med. Hyg. 54, 632 – 642. Felger, I., Livingstone, T., Kabintik, S., Marshall, V., Genton, B., Alpers, M., Beck, H.P., 1994. Plasmodium falciparum: extensive polymorphism in merozoite surface antigen 2 alleles in an area with endemic malaria in Papua New Guinea. Exp. Parasitol. 79, 106 – 116. Felger, I., Irion, A., Steiger, S., Beck, H.P., 1999. Genotypes of merozoite surface protein 2 in Tanzania. Trans. R. Soc. Trop. Med. Hyg. 93, Suppl. 1. S1/3 – S1/9. Foley, M., Randford-Cartwright, L.C., Babiker, H.A., 1992. Rapid and simple method for isolating malaria DNA from fingerprick samples of blood. Mol. Biochem. Parasitol. 53, 241 – 244. Snounou, G., Viriyakosol, S., Zhu, X.P., Jarra, W., Pinheiro, L., do Rosario, V.E., Thaithong, S., Brown, K.N., 1993. High sensitivity of detection of human malaria parasite by the use of nested polymerase chain reaction. Mol. Biochem. Parasitol. 61, 315 – 320.
.
Rapid DNA extraction for molecular epidemiological studies of malaria Lars Henning a,b, Ingrid Felger a, Hans-Peter Beck a,* b
a Swiss Tropical Institute, Socinstrasse 57, 4002 Basel, Switzerland Uni6ersity of Witten-Herdecke, Stockumerstrasse 10, D-58448 Witten, Germany
Received 20 May 1998; received in revised form 8 October 1998; accepted 19 October 1998
Abstract DNA isolation from blood samples collected in molecular epidemiological studies is crucial for the quality and reproducibility of data. Blood samples from two malaria endemic sites have been prepared by four different DNA isolation methods with subsequent PCR amplification of the msp2 locus of Plasmodium falciparum. We tested a rapid boiling method; the guanadine isothiocyanate DNA extraction; QIAmp™ blood kit; and the ISOCODE™ STIX PCR template preparation dipstick, and analysed the numbers of concurrent infections/sample. The rapid boiling method and the ISOCODE™ STIX provided overall the best sensitivity combined with ease of handling. The possibility to store and ship the ISOCODE™ STIX at ambient temperature adds further advantage to this method. © 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: DNA extraction; Molecular epidemiology; Malaria; Polymerase chain reaction; msp2 ; Genotyping; Plasmodium falciparum
Abbre6iations: GTC, guanidine isothiocyanate; MSP 2, merozoite surface protein 2; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism. * Corresponding author. Tel.: + 41-61-284-8116; fax: + 41-61-271-8654; e-mail: [email protected]. 0001-706X/99/$ - see front matter © 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 9 8 ) 0 0 0 9 0 - 4
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1. Introduction Over the past few years PCR-based genotyping of pathogens has become a central technique applied in diagnostics and in molecular epidemiological studies. PCR amplification of a marker sequence is a convenient and practical tool in epidemiological studies, where only limited amounts of specimen, such as blood, are available. The extraction of DNA from such samples is a critical step and several methods are being used. In malaria field studies involving detection of parasite DNA and genotyping of the infecting parasites, mostly finger prick blood samples are collected (Felger et al., 1994; Beck et al., 1997). DNA is routinely isolated from these samples either by phenol/chloroform extraction or by a rapid boiling method (Foley et al., 1992). Methods for DNA preparations from blood samples in studies of epidemiological scale have to fulfil the following criteria: (1) rapid preparation and large through put, (2) high reliability, (3) production of DNA of good quality for long-term storage, (4) avoidance of cross-contamination, (5) reasonable costs. In this study we compared four different methods of DNA preparation from samples collected during malaria field studies in order to test for their reliability, sensitivity, and ease of handling. The following methods were compared: rapid boiling method; guanidine isothiocyanate (GTC) preparation with subsequent phenol/chloroform extraction; a commercially available DNA purification kit (QIAamp® Blood Kit Cat.No.29104, Qiagen, Basle, Switzerland); and a new dipstick for DNA preparation (ISOCODE™ STIX PCR Template Preparation Dipstick, Schleicher&Schuell). The ISOCODE™ dipstick also allows storage and transportation at ambient temperature and thus was expected to be a practical tool for field studies. Because of this advantage for field studies we compared DNA obtained from the ISOCODE™ STIX for PCR amplification with DNA prepared by the other three standard extraction methods in order to assess reliability and sensitivity. DNAs prepared by these different techniques were compared in a standard PCR amplification of the merozoite surface protein (MSP2) locus of Plasmodium falciparum. msp2 has been frequently used in genotyping studies as highly polymorphic marker (Felger et al., 1994; Contamin et al., 1996). In blood samples from areas of high malaria endemicity, multiple P. falciparum infections have been frequently observed (Felger et al., 1994). As a measurement for sensitivity we used the number of msp2 genotypes detected after PCR amplification of templates prepared by the different extraction methods. Because of the lack of fresh infected whole blood, DNA was extracted from 40 pellets of packed cells which had been stored at −20°C. Red blood cells were diluted with serum-free RPMI in order to reconstitute the red blood cell concentration. Packed cells were derived from blood samples collected in Papua New Guinea (10) and Tanzania (30) during two field studies. Parasitaemia levels within the 40 samples ranged from 1 to 739 parasites/200 leucocytes. Nine samples were microscopy negative.
L. Henning et al. / Acta Tropica 72 (1999) 149–155
151
2. Template preparation
2.1. QIAamp ® Blood Kit (Cat.No.29104, Qiagen, Basle, Switzerland) The kit was used according to the supplier’s instruction. Briefly, 10 ml of blood were diluted in 190 ml phosphate-buffered saline (PBS). Two hundred microlitres of Buffer AL and 25 ml of the provided Proteinase K were added and immediately vortexed for 15 s. After incubating for 10 min at 70°C, DNA was precipitated with 210 ml 96% ethanol. The mixture was applied to a QIAamp® spin column and after centrifugation, the column was washed twice with Buffer AW. DNA was eluted with 50 ml dH2O at 70°C. Five microlitres of this DNA solution were used as template for PCR amplification. Affinity purification yields good quality DNA, but is comparatively expensive.
2.2. Rapid boiling method Ten microlitres of blood were added to 500 ml ice-cold 5 mM sodium phosphate buffer (pH 8.0), vortexed and spun for 2 min at 14 000 × g. The supernatant was discarded and washing was repeated twice. Fifty microlitres dH2O were added to the washed pellet, and after resuspension, boiled for 10 min. Finally, the sample was spun for 10 min at 14 000×g and the pellet discarded. Five microlitres of the supernatant were used for PCR amplification. This method is very fast and inexpensive, nevertheless long-term storage affects the quality of the DNA isolated by this method.
2.3. GTC/phenol/chloroform extraction Fifty microlitres GTC (4 M guanidine isothiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sodium sarcosylsufate) and 5 ml of 2 M sodium acetate (pH 4.4) were added to 10 ml of blood. Subsequently, 50 ml phenol (pH 8, equilibrated with Ph-Maker™, Oncor-Appligene, France) and 50 ml chloroform were added, the mixture was vortexed and placed on ice for 15 min. The sample was spun for 10 min at 14 000 ×g, the aqueous phase transferred to a new tube and precipitated with 100 ml isopropanol. After centrifugation for 20 min at 14 000× g, the supernatant was discarded and the pellet washed with 200 ml 70% ethanol and spun again. The air-dried pellet was dissolved in 50 ml dH2O and 5 ml were used for PCR amplification. This method is time consuming and prone to contamination due to frequent pipetting steps and changes of tubes.
2.4. ISOCODE™ STIX PCR Template Preparation Dipstick (Cat.No. 495020, Schleicher & Schuell, Dassel, Germany) DNA was extracted from the dipstick according to the supplier’s instructions. Briefly, 10 ml of blood were loaded onto one triangle of the dipstick and dried for 3 h at room temperature. The triangle was clipped off and transferred into a
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L. Henning et al. / Acta Tropica 72 (1999) 149–155
microfuge tube and washed twice with 500 ml dH2O by vortexing three times for at least 5 s. Vortexing less than 5 s may lead to DNA of lower quality impeding subsequent amplification. After complete removal of dH2O, the tube was spun for 5 s and residual water was pipetted off. Fifty microlitres of dH2O were added and incubated for 30 min at 95°C. Finally, the tube was gently tapped 20 times and the supernatant transferred into a new tube. PCR amplification was performed with 5 ml DNA solution. We also added the complete triangle after two washes into the PCR-reaction with similar results or with increased sensitivity.
2.5. PCR amplification PCR amplification of the msp2 locus was performed as described previously (Felger et al., 1994). In all experiments, 5 ml DNA template were used in the primary PCR reaction and 2 ml primary product were used in a 100 ml nested PCR reaction using a Perkin Elmer DNA Thermal Cycler 480. Ten microlitres of nested product were loaded onto a 10% polyacrylamide gel, run, stained with ethidium bromide, and visualized by UV-illumination.
2.6. Genotyping Fifteen microlitres nested PCR product were digested with Hinf I (Biolabs, New England), producing a genotype-specific RFLP-pattern (Felger et al., 1999). For higher resolution and discrimination power, e.g. in the case of a high number of concurrent infections, additional Dde I-, Rsa I-, and ScrF I-digests (all Biolabs) were performed. Restriction fragments were separated on a 10% polyacrylamide gel. For determination of fragment lengths a 1 kb DNA ladder (Gibco-Lifetechnologies, Basle, Switzerland) was used. For quality control, primary and nested PCR from 10% of the samples was repeated and negative samples were all repeated once with equal results. The analysis was carried out by two persons independently. A full comparison of 40 blood samples, including samples negative and positive for P. falciparum by microscopy, was performed using DNA purified with QIAamp® blood kit and ISOCODE™ STIX. The numbers of concurrent infections (multiplicity) defined by msp2 genotyping in these 40 samples are listed in Table 1. The average number of genotypes found per sample with ISOCODE™ STIX preparation was higher than with the QIAamp® preparation. However, this difference was not statistically significant. Nineteen samples had equal multiplicity when the amplification results of the two extraction methods were compared. With the applied RFLP typing scheme, msp2 alleles can be classified into two distinct allelic families: FC27 and 3D7. There was no difference in the detection sensitivity for both families with both template preparations. Fig. 1 depicts the correlation between numbers of alleles found by either method. There were five samples found negative with QIAamp® prepared template, and which were positive using ISOCODE™ STIX prepared template. Two of these were negative by microscopy, the remaining three had low parasite densities (53 parasites/200 leucocytes).
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153
A subset of 12 samples were further used to compare template preparation with GTC/phenol/chloroform extraction and with the rapid boiling method. Table 2 shows the mean number of msp2 alleles/sample detected using templates prepared with all four DNA preparations. Differences found were not statistically significant. Furthermore, the number of alleles detected in each sample was used to establish a ranking for the sensitivity of the four methods. Six out of 12 samples tested had an equal number detected by all four methods. The rapid boiling method and the ISOCODE™ STIX provided the best result in four samples. Within the remaining two samples either QIAamp® or GTC /phenol/chloroform extraction gave the best results on their own. Hence, using the rapid boiling method or the ISOCODE™ STIX method provides the most sensitive determination of overall multiplicity. In order to determine the detection limit of the ISOCODE™ STIX preparation method, serial dilutions of packed cells from blood with a parasitaemia of 5% were made in PBS starting from 10 ml of packed cells. The detection limit for msp2 PCR amplification was calculated on the assumption that 1% parasitaemia corresponds to 50 000 parasites/ml blood. The greatest dilution of the template which still produced an amplification product using 10 ml of template was 1:100 000, which corresponds to 2.5 parasites/ml blood (parasitaemia of 0.00005%). This is in a comparable range of sensitivity reported by others (Foley et al., 1992; Snounou et al., 1993). However, the detection limit by PCR varies depending on the specific gene chosen for amplification. Binding capacity of the ISOCODE™ STIX was also examined. Therefore, the sample application area of the dipstick was loaded with 4, 6, 8, 10, 12, 15, 20, 25 ml whole blood from a single blood sample, respectively. The amount of loaded blood did not affect the detection, and one genotype was detected throughout all samples. Furthermore, storage of template prepared using the ISOCODE™ STIX for 2 months at + 4°C did not affect the template quality and equal results were obtained when tested on four positive samples. Our study showed that template preparation by the rapid boiling method or using the ISOCODE™ STIX provided increased sensitivity for the detection of multiple infections when compared to QIAamp® or GTC/phenol/chloroform template preparation. Although the rapid boiling method offers high sensitivity and Table 1 Comparison of two different DNA extraction methods with respect to the number of msp2 alleles detected in 40 samplesa
Mean multiplicity ( 9 SE) Mean FC27 multiplicity ( 9 SE) Mean 3D7 multiplicity ( 9 SE) Positive samples Higher multiplicity found by the respective method a
QIAamp®
ISOCODE™ STIX
2.38 9 0.33* 1.23 90.18* 1.15 9 0.17* 28 8/21
2.58 9 0.33* 1.2390.17* 1.28 9 0.20* 33 13/21
Multiplicity, number of genotypes detected per blood sample; SE, standard error. * Difference not significant.
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L. Henning et al. / Acta Tropica 72 (1999) 149–155
Fig. 1. Comparison of number of alleles found by ISOCODE™ STIX and QIAamp® in 40 samples.
cost-effectiveness, long-term storage of template is critical. The ISOCODE™ STIX offers good sensitivity and in addition, convenient sample handling and circumvents the need for transportation of blood samples using a cold chain. We have carried out studies in Papua New Guinea, Sao Tome and Tanzania successfully using the ISOCODE™ STIX, which was tested by also using frozen EDTA-blood. The DNA preparation is fast, taking less than 1 h, and is easily performed. The prepared DNA was shown to be of high quality and allows long-term storage. Furthermore, minimal handling steps minimize the risk of cross-contamination. The good performance and the additional advantages of the ISOCODE™ STIX seem to make it a reliable tool for molecular epidemiological studies involving PCR and would offer a convenient method to standardize such genotyping studies, which is urgently needed.
L. Henning et al. / Acta Tropica 72 (1999) 149–155
155
Table 2 Mean multiplicity of infections detected by four different DNA extraction methods Extraction method
Sample size
Total alleles
Mean multiplicity*
Rapid boiling method GTC/Phenol/Chloroform QIAamp® ISOCODE™ STIX
12 12 12 12
26 21 19 24
2.17 90.57 1.75 90.53 1.58 90.52 2.00 90.59
* Differences between the methods were not statistically significant.
Acknowledgements Lars Henning was supported by the Daniela und Ju¨rgen Westphal-Stiftung. This work obtained financial support from the German Science Foundation (DFG) grant no. BE1075/2-1.
References Beck, H.P., Felger, I., Huber, W., Steiger, S., Smith, T., Weiss, N., Alonso, P., Tanner, M., 1997. Analysis of multiple Plasmodium falciparum infections in Tanzanian children during the phase III trail of the malaria vaccine SPf66. J. Infect. Dis. 175, 921 – 926. Contamin, H., Fandeur, T., Rogier, C., Bonnefoy, S., Konate, L., Trape, J.F., Mercereau-Puijalon, O., 1996. Different genetic characteristics of Plasmodium falciparum isolates collected during successive clinical malaria episodes in Senegales children. Am. J. Trop. Med. Hyg. 54, 632 – 642. Felger, I., Livingstone, T., Kabintik, S., Marshall, V., Genton, B., Alpers, M., Beck, H.P., 1994. Plasmodium falciparum: extensive polymorphism in merozoite surface antigen 2 alleles in an area with endemic malaria in Papua New Guinea. Exp. Parasitol. 79, 106 – 116. Felger, I., Irion, A., Steiger, S., Beck, H.P., 1999. Genotypes of merozoite surface protein 2 in Tanzania. Trans. R. Soc. Trop. Med. Hyg. 93, Suppl. 1. S1/3 – S1/9. Foley, M., Randford-Cartwright, L.C., Babiker, H.A., 1992. Rapid and simple method for isolating malaria DNA from fingerprick samples of blood. Mol. Biochem. Parasitol. 53, 241 – 244. Snounou, G., Viriyakosol, S., Zhu, X.P., Jarra, W., Pinheiro, L., do Rosario, V.E., Thaithong, S., Brown, K.N., 1993. High sensitivity of detection of human malaria parasite by the use of nested polymerase chain reaction. Mol. Biochem. Parasitol. 61, 315 – 320.
.