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ORIGINAL ARTICLE
Year : 2016  |  Volume : 9  |  Issue : 1  |  Page : 13-17

Assessment of diagnostic performance of a commercial direct blood PCR kit for the detection of Schistosoma mansoni infection in mice compared with the pre-extracted PCR assay


Parasitology Department, Faculty of Medicine, Benha University, Benha, Egypt

Date of Submission20-Dec-2015
Date of Acceptance29-Feb-2016
Date of Web Publication25-Oct-2016

Correspondence Address:
Maysa A Eraky
Parasitology Department, Faculty of Medicine, Benha University, Benha 13518
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-7942.192995

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  Abstract 

Background
Different diagnostic techniques have been used in the diagnosis of schistosomiasis. However, they are far from ideal regarding its early diagnosis. PCR techniques have been tried to improve the direct detection of schistosomiasis.
Objective
The aim of the present study was to evaluate the diagnostic performance of direct amplification of Schistosoma mansoni DNA in the early prepatent period in experimentally infected mice by PCR technique using unextracted DNA as PCR template compared with pre-extracted S. mansoni DNA samples.
Materials and methods
Mice were infected by 100±10 S. mansoni cercariae. Three mice were sacrificed every 3 or 4 days for 5 weeks. Whole blood samples were collected for direct amplification without prior extraction. Serum samples were pooled, and the extracted DNA was detected by using the KAPA blood PCR kit and conventional PCR methods. The diagnostic performance was compared between the two methods.
Results
The results showed that the diagnosis of S. mansoni utilizing pre-extracted DNA was superior to direct amplification of DNA, bypassing nucleic acid extraction which failed to detect S. mansoni DNA in any of the examined samples. Pre-extracted DNA was detected in all samples from the second day after infection by using the two PCR techniques.
Conclusion
These results indicate that S. mansoni infection cannot be efficiently detected directly by using the PCR technique without pre-extraction of DNA from whole blood samples using the KAPA blood PCR kit.

Keywords: direct PCR amplification, early diagnosis, Schistosoma mansoni, whole blood


How to cite this article:
Eraky MA, Aly NS. Assessment of diagnostic performance of a commercial direct blood PCR kit for the detection of Schistosoma mansoni infection in mice compared with the pre-extracted PCR assay. Parasitol United J 2016;9:13-7

How to cite this URL:
Eraky MA, Aly NS. Assessment of diagnostic performance of a commercial direct blood PCR kit for the detection of Schistosoma mansoni infection in mice compared with the pre-extracted PCR assay. Parasitol United J [serial online] 2016 [cited 2020 Sep 30];9:13-7. Available from: http://www.new.puj.eg.net/text.asp?2016/9/1/13/192995


  Introduction Top


Chronic schistosomiasis may lead to an increase in the mortality and morbidity rates due to egg deposition followed by granuloma formation in different organs. Thus, a diagnostic tool able to detect schistosomiasis in its early acute phase would be of great value, allowing for early treatment and prevention of egg-induced irreversible pathological reactions associated with chronic infections [1]. In highly endemic areas, where transmission rates are high, ovum detection after mass drug administration becomes inaccurate [2]. The routine confirmation of schistosomiasis is done through microscopic detection of eggs in patient stool samples. However, sensitivity of this diagnostic method is low for detecting light infections and infection rates in low transmission areas. Efficient, rapid, and reproducible procedures are required for the diagnosis of early schistosomiasis.

The use of PCR technology employing specific DNA as the essential substrate has proved to be a breakthrough in clinical diagnosis. Various different inhibitors connected to clinical and environmental factors can affect PCR sensitivity and accurate pathogen detection. PCR inhibition interferes with cell lysis, and sequestration or degradation of nucleic acids, and hinders the polymerase activity [3]. In blood, heme blocks active polymerase sites [4], and proteases degrade the polymerase. Other inhibitors include complex polysaccharides [5], bilirubin and bile salts found in stool [6], as well as humid compounds in soils and sediments [7]. Options for controlling PCR inhibition include sample manipulation or decreasing the amount so as to remove or dilute inherent matrix-derived inhibitors [8],[9],[10]. Processing a sample before PCR detection is important for the exclusion of inhibitory factors, as well as concentration of the target template and release of the nucleic acid in purified form. Therefore, the standard protocol is to include a DNA purification step. However, the established procedures for DNA extraction are time-consuming and costly; besides there exists a possibility of DNA loss during sample extraction, which changes the quality of the DNA profile obtained. Accordingly, techniques of DNA extraction are not completely efficient, and the resultant nucleic acids from sample processing are always less than the original nucleic acid input inhibitors [8]. This is especially important when the targeted nucleic acids are originally present at low concentrations, as is the case in most clinical and environmental samples. In addition, some inhibitors may be eluted following purification, thus producing false negative results. This has been observed as false-negative PCR results after several different types of purification methods for the detection of hepatitis B virus (HBV) [11]. As a result of false negative results, treatment and control, especially for highly pathogenic agents, are delayed. Direct PCR assessment without sample processing presents a promising solution [12]. PCR protocols that allow direct amplification without DNA purification have been reported in the literature [13].

In their study, Hamburger et al. [14] described the presence of a 121-base pair tandem repeat DNA sequence in 12% of S. mansoni genome using specific primers previously validated in a study by Pontes et al. [15]. The latter researchers reported, for the first time, the high sensitivity of the PCR technique in the detection of free-circulating S. mansoni DNA in the serum of two patients with active intestinal schistosomiasis, and its specificity having no cross-reaction with DNA from other helminths. Moreover, Sadek et al. [16] used the primers that were used by Pontes et al. [15] in the diagnosis of active intestinal schistosomiasis and detected circulating DNA of S. mansoni with a sensitivity of 97.2% and a specificity of 100%. In another important study, Hussein et al. [1] confirmed that these primers could provide high sensitivity and specificity for the detection of S. mansoni in the early prepatent period.

In spite of the urgent need for affordable and easy methods for testing and monitoring of S. mansoni infection, there is no study in the literature on the role of direct PCR for evaluating patients infected with S. mansoni in Egypt, where schistosomiasis is endemic and an important health problem. Consequently, the present study was designed to evaluate the feasibility of direct PCR for direct amplification of S. mansoni DNA, bypassing the nucleic acid extraction. Results obtained from direct PCR were compared with those obtained by routine molecular techniques utilizing whole blood and serum samples.


  Materials and methods Top


Type of study

This was a controlled experimental study.

Study design

Experimental S. mansoni infection

In total, 30 Swiss albino female mice 7 weeks old and weighing 20–22 g were used in the present study. The animals were maintained under standard conditions in the Experimental Research Center of Theodor Bilharz Research Institute, Cairo, Egypt. Mice were infected transcutaneously by exposing them to 100±10 S. mansoni cercariae (Egyptian strain)/mouse. Every 3 or 4 days, through the first 5 weeks after infection, three mice were randomly chosen from the infected animals, and blood was collected through cardiac puncture. Serum of each mouse was separated by centrifugation (3000 rpm for 15 min). Half milliliter blood was collected from each mouse and emptied immediately in a vacutainer tube containing EDTA. Collected sera and whole EDTA blood from each of the three mice in the same time period were pooled and used for PCR. All collected samples were kept frozen at −80°C until use. S. mansoni eggs were obtained from the livers of Swiss albino mice 8 weeks after infection, suspended in 0.9% saline, and stored at −20°C until use [17]. The number of eggs in 0.9% saline suspension was quantified using a Neubauer chamber, and a solution containing ∼2000 eggs was subjected to DNA extraction, to be used as a positive control [15].

Extraction of S. mansoni DNA from eggs and serum samples

S. mansoni DNA was extracted from each pooled serum sample (200 μl) using a QIAamp mini DNA extraction kit (Qiagen GmbH, Germany) according to manufacturer’s instructions for automatic extraction in a QIAcube extractor (Qiagen GmbH). The extracted DNA concentration was confirmed by measurement using a UV spectrophotometer. Readings were taken at wavelengths of 260 and 280 nm, as reported in a study by Alhusseini et al. [18]. Concentration of DNA sample was measured as 50 μg/ml×A260×dilution factor [19].

Amplification of S. mansoni DNA by PCR

DNA samples were amplified using the forward 5′-GATCTGAATCCGACCAACCG-3′ and reverse 5′-ATATTAACGCCCACGCTCTC-3′ primers, designed by Pontes et al. [15], to generate a 110-bp product following the amplification procedure. These primers were designed to amplify the 121-bp tandem repeat DNA sequence of S. mansoni, as described by Hamburger et al. [14]. For serum amplification, 2 μl of undiluted extracted DNA was used as a template. All reactions were carried out in a rapid cycler PCR (G-Storm Thermal cycler; Applied Biosystems, Foster City, California, USA; England) using a 10-μl mixture containing 20 mmol/l Tris-HCl (pH 8.4), 50 mmol/l KCl buffer, 1.5 mmol/l MgCl2, 0.5 μmol/l of each primer, 200 μmol/l dNTPs, and 0.75 U of Taq DNA polymerase. The two-step amplification cycle consisted of denaturation at 95°C for 45 s and then an annealing step at 63°C for 30 s. A total of 40 cycles were carried out. The denaturation step was prolonged for 5 min in the first cycle, and the last cycle included an extension step of 2 min.

Direct amplification of S. mansoni DNA without nucleic acid extraction

Using the same primer pair for S. mansoni, direct amplification was carried out for each of the EDTA blood samples using the KAPA blood PCR kit (Kapa Biosystems, Inc., USA).

The reaction mix was prepared using 2.5 μl EDTA blood per 25 μl rxn (per reaction volume), 25 μl of KAPA PCR Mix B (2×) (containing all PCR components, except the primers and template), and 2.50 μl of each primer. Then, PCR grade water was added to each tube and the volume was adjusted to 50 μl. The tubes were vortexed before placing them in a thermocycler. Amplification was carried out in a rapid cycler PCR (G Storm Thermal cycler). According to the manufacturer’s instructions, the applied amplification programme included initial denaturation at 95°C for 5 min and then cycling for 40 cycles (95°C for 30 s, 56°C for 30 s, and 72°C for 1 min). The final extension was at 72°C for 2 min. Positive and negative controls were used for each run.

Confirmation of direct amplification KAPA blood PCR validity

The validity of the direct amplification KAPA Blood PCR kit was tested in two ways: first, by using positive HBV samples, which, in a study by Alhusseini et al. [20], was subjected to direct amplification by using the same kit, giving promising positive results; and, second, by amplification of the pre-extracted DNA.

Post-PCR processing and analysis of the amplified products

After completion of the PCR programme, the obtained amplified products were centrifuged at a speed of 14000 rpm to obtain the most compact pellet of organic debris and to facilitate recovery of the amplicon-containing supernatant. The amplified DNA was analyzed through electrophoresis. About 10 μl of each reaction mixture and 1000-bp ladder (molecular weight marker) were separated on 2% agarose gel containing 0.3 μg/ml ethidium bromide. The bands were visualized using an UV transilluminator (254 nm) and photographed using an 8 megapixel digital camera. The image was transferred to be analyzed on a computer software (Alpha InnoTech Gel Documentation System).

Ethical considerations

The present study was approved by the Ethical Committee of Faculty of Medicine, Benha University, Egypt (2015). All the animals were handled in strict accordance to the good animal practice as defined by the Animals Use Ethics Committee of Faculty of Medicine, Benha University, Egypt.


  Results Top


The results of the present study showed that direct KAPA blood PCR kit failed to detected S. mansoni DNA in any of the tested samples, as all the examined samples gave negative results ([Figure 1], lanes 1–6), whereas those preceded by DNA extraction and then examined using the same kit gave positive bands at 110 bp using gel electrophoresis ([Figure 1], lanes 7–11). All samples tested using conventional PCR technique (preceded by an amplification step) gave positive results and S. mansoni DNA was detected from the second day after infection ([Figure 2]).
Figure 1 Gel electrophoresis of the amplified products of S. mansoni DNA; M: 1000 bp marker. Lanes 1–6: negative for unextracted S. mansoni DNA amplification using the KAPA blood PCR kit. Lanes 7–11: positive for pre-extracted S. mansoni DNA amplification using the KAPA blood PCR kit.

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Figure 2 Gel electrophoresis of the amplified products of pre-extracted S. mansoni DNA. M: 1000 bp marker. Lane 1: positive control. Lanes 2, 3, 4, 5: positive for pre-extracted S. mansoni DNA using the conventional PCR method.

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  Discussion Top


Direct PCR technology facilitates PCR amplification directly from small amount of samples without DNA extraction and purification. It eliminates any sample pre-treatment steps, and thus is very useful to save time and cost and protect samples from cross-contamination or loss when dealing with multiple tissues or samples by eliminating tedious genomic DNA preparation. The availability of PCR results within several hours of taking samples allows the clinicians to adapt their treatment very rapidly with a potential positive effect on the final prognosis.

The present controlled study was an attempt to determine the performance of the commercially available direct KAPA blood PCR kit as an alternative to the readily used conventional pre-extraction PCR procedure in the early detection of S. mansoni DNA in the prepatent period in experimentally infected mice. This kit has been validated for direct amplification from crude sample types, including fresh or frozen whole blood, blood collected in EDTA tubes, or on FTA® Elute Cards, ‘Guthrie cards’, or filter paper. Overall, 10% sample concentration (10%) was tested in comparison with the pre-extracted DNA samples using the direct and conventional PCR kits, respectively.

Before carrying out the direct PCR technique, the direct amplification kit was tested to ensure its validity using previously confirmed HBV-positive samples. HBV DNA was previously amplified in a study by Alhusseini et al. [20] using the same KAPA blood PCR kit. The same results were obtained using our reagents (kit), and the virus DNA was also successfully amplified directly without need for prior DNA extraction. In addition, pre-extracted S. mansoni DNA was also successfully amplified ([Figure 1]) by using the direct amplification kit.

Regarding the diagnostic methods used in our study for the detection of S. mansoni DNA in serum and whole EDTA blood samples of experimentally infected mice, it was found that pre-extracted DNA was superior to direct amplification of DNA, bypassing nucleic acid extraction which failed to detect S. mansoni DNA from any of the examined samples ([Figure 1]), whereas pre-extracted DNA was detected in all samples from the second day after infection ([Figure 1] and [Figure 2]).

This failure may be due to the inability of the tested technique to detect S. mansoni DNA in the utilized samples; however, this is not logical, as the kit efficiently amplified the same target gene in all the pre-extract samples. Another possible explanation of this failure is the presence of inhibitors in the samples (hemoglobin), interfering with the direct amplification. Other factors include unsuitable sample size, whereas a large sample size of whole blood may turn out to be detrimental to the PCR reaction [21]. Heme present in blood can block the polymerase’s active site, and proteases can degrade the polymerase [4].

There is a striking difference in the blood-resistant performance between several direct PCR kits due to the mutational alteration of DNA polymerases to render them resistant to inhibition by blood components. Miura et al. [22] compared the PCR performance of six commercially available direct PCR-type DNA polymerases with a standard Taq DNA polymerase in the presence of PCR inhibitors found in blood components using a diagnostic nested PCR method for the detection of Plasmodium spp. genomic DNA. They found that KOD FX and BIOTAQ DNA polymerases were resistant to the inhibitory effects of blood components in 40% blood eluent reaction mixtures, whereas KAPA blood DNA polymerase was the least as it was resistant to the inhibitory effects of blood components in 10% blood eluent reaction mixtures.

The current study was the first one to evaluate the diagnostic performance of direct amplification of S. mansoni DNA. In addition, in the field of parasitology, few studies have been conducted. Using the same KAPA blood PCR kit, El-Sayed et al. [23] successfully amplified Acanthamoeba DNA directly from corneal scrap, bypassing nucleic acid extraction. They found that direct amplification of Acanthamoeba DNA was superior to microscopy and culture. Their results were in agreement with those of Zhao et al. [24], who found that the direct PCR assay without template DNA extraction using specific primers for fungi, bacteria, herpes simplex virus-1, and Acanthamoeba is a rapid diagnostic technique with high sensitivity and specificity.


  Conclusion Top


From the results of the present study, it was concluded that conventional PCR was superior to direct PCR in the early detection of S. mansoni DNA using KAPA Blood PCR kit, which proved to be an unreliable method for direct molecular diagnosis of schistosomiasis at 10% sample concentration. Due to the advantages of direct PCR, it is recommended to conduct further analysis using different sample concentrations and to try other commercially available direct PCR amplification kits.

Authors contribution

MA Eraky conceived and designed the study, performed the experiments, analyzed the data and wrote the manuscript. NSM Aly performed the experiments, analyzed the data and revised the manuscript.

Both authors of declare that the manuscript has been read and approved by them that the requirements for authorship as stated earlier in this document have been met, and that each author believes that the manuscript represents honest work.

Acknowledgement

The authors are grateful to Assistant Professor Naglaa Fathy Alhusseini, Department of Biochemistry, Faculty of Medicine, Benha University, Egypt, for her technical advice.

Author contribution

MA Eraky conceived and designed the study, performed the experiments, analyzed the data and wrote. NSM Aly performed the experiments, analyzedthe data and revised.

Both authors of declare that the manuscript has been read and approved by all the authors, that the requirements for authorship as stated earlier in this document have been met, and that each author believes that the manuscript represents honest work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2]


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