Storage of viruses on filter paper for genetic analysis

Pitcovski, Jacob; Shmueli, E.; Krispel, S; Levi, Nehora

doi:10.1016/s0166-0934(99)00101-9

Cited by 22 publications

(15 citation statements)

References 21 publications

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“…While the usefulness of filter paper blood samples for the measurement of MV-specific serum antibodies had been demonstrated before (3,12), recent publications have suggested that filter paper blood samples may also be suitable for RT-PCR analyses to diagnose certain virus infections (1,15). In the present study we show that blood samples spotted on filter paper are indeed suitable for use in MV-specific RT-PCR analysis.…”

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confidence: 60%

Combination of Reverse Transcriptase PCR Analysis and Immunoglobulin M Detection on Filter Paper Blood Samples Allows Diagnostic and Epidemiological Studies of Measles

et al. 2001

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As measles control and elimination campaigns progress, laboratory confirmation of clinically diagnosed measles cases becomes increasingly important. However, in many tropical countries collection and storage of clinical specimens for this purpose are logistically complicated. In this study it is shown that blood samples spotted on filter paper are suitable for the laboratory diagnosis of measles using a combination of reverse transcriptase PCR (RT-PCR) analysis and immunoglobulin M (IgM) detection. First, it was shown that in vitro measles virus (MV)-infected cells diluted in human blood and spotted on filter paper can be detected by RT-PCR. Small amounts of infected cells remained detectable after 25 weeks of storage of the filter paper at room temperature, 4 weeks at 37°C, or 2 weeks at 45°C. Subsequently, this RT-PCR was applied to filter paper blood samples collected from 117 clinically diagnosed measles patients in Sudan in 1997 and 1998. Prior laboratory diagnosis had confirmed 90 cases as acute MV infections, while 27 proved to be nonmeasles rash disease cases. Positive RT-PCR signals were detected in filter paper blood samples of 43 of the 90 confirmed cases (48%) but in none of the 27 nonmeasles cases. In addition, MV-specific IgM levels measured in reconstituted filter paper samples correlated well with those measured in plasma samples. Measles diagnosis based on the combination of filter paper RT-PCR and IgM detection had a sensitivity and specificity of 99 and 96%, respectively. An advantage of this diagnostic approach is that sequencing of RT-PCR products allows phylogenetic analysis of the MV strain involved.

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confidence: 60%

Combination of Reverse Transcriptase PCR Analysis and Immunoglobulin M Detection on Filter Paper Blood Samples Allows Diagnostic and Epidemiological Studies of Measles

et al. 2001

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“…However, since no stored serum samples were available, we used smears of serially collected bone marrow samples, which are routinely obtained during treatment (from four to six bone marrow samples were available for each patient). This approach was chosen based on recent reports (1,23) of the potential of reverse transcription (RT)-PCR to detect viral RNA in dried blood spots. We thus attempted to investigate the presence of HCV RNA in glass slide preparations of bone marrow aspirates which had been serially collected from the five children involved in the outbreak and which had been stored in sterile sealed bags at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Use of the Minimum Spanning Tree Model for Molecular Epidemiological Investigation of a Nosocomial Outbreak of Hepatitis C Virus Infection

et al. 2004

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The minimum spanning tree (MST) model was applied to identify the history of transmission of hepatitis C virus (HCV) infection in an outbreak involving five children attending a pediatric oncology-hematology outpatient ward between 1992 and 2000. We collected blood samples from all children attending since 1992, all household contacts, and one health care worker positive for antibody to HCV (anti-HCV). HCV RNA detection was performed with these samples and with smears of routinely collected bone marrow samples. For all isolates, we performed sequence analysis and phylogenetic tree analysis of hypervariable region 1 of the E2 gene. The MST model was applied to clinical-epidemiological and molecular data. No additional cases were detected. All children, but not the health care worker, showed genotype 3a. On six occasions, all but one child had shared the medication room with another patient who later seroconverted. HCV RNA detection in bone marrow smears revealed, in some cases, a delay of several months in anti-HCV responses. Sequence analysis and phylogenetic tree analysis revealed a high identity among the isolates. The MST model applied to molecular data, together with the clinical-epidemiological data, allowed us to identify the source of the outbreak and the most probable patient-to-patient chain of transmission. The management of central venous catheters was suspected to be the probable route of transmission. In conclusion, the MST model, supported by an exhaustive clinical-epidemiological investigation, appears to be a useful tool in tracing the history of transmission in outbreaks of HCV infection.Since the introduction of blood donor screening through antibody testing, the risk of acquiring infection with hepatitis C virus (HCV) through the transfusion of blood or blood products has dramatically decreased in industrialized countries (8,28,34). Nonetheless, the nosocomial transmission of HCV continues to occur (21). Several recent studies provided evidence of patient-to-patient transmission in most of the cases of nosocomial transmission (2, 5, 6, 9, 13, 15-17, 19, 20, 29, 36), although some studies also reported transmission from health care workers to patients and vice versa (10,26,32).In recent years, the use of molecular biology techniques has proven to be a powerful tool in the epidemiological investigation of HCV infection in health care facilities and other settings (2,4,6,9,10,13,15,16,19,20,24,30,32). In particular, phylogenetic tree analysis has often been used to identify the original source of infection. However, this technique does not allow a detailed history of transmission to be traced or the modes of transmission to be identified, except in cases of provider-to-patient transmission.In an attempt to more completely describe nosocomial outbreaks of HCV infection, we adopted a parsimonious theoretical approach, referred to as the minimum spanning tree (MST) model (see Appendix). The MST approach, a concept of the graph theory, represents one of the most common problems of combinatorial optim...

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“…Washing filter papers has reduced the amount of inhibitors, which in turn has allowed for conventional PCR on the filter disk (25). Alternatively, DNA samples have been extracted and/or purified from specimens on filters and used for PCR (3,5,18,27). Although dried blood spots can be used for CMV screening (2), the assay may not detect some cases of congenital infection, as blood specimens contain smaller amounts of CMV than urine specimens (4, 11; N. Inoue and S. Koyano, unpublished results).…”

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Real-Time PCR Assay Using Specimens on Filter Disks as a Template for Detection of Cytomegalovirus in Urine

et al. 2007

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Since congenital cytomegalovirus (CMV) infection causes late-onset sequelae, the identification of CMVinfected newborns is important. For this purpose, we established a simple real-time PCR assay using a filter disk. Combined with the collection of urine using filter papers placed in the diaper, this assay can make CMV screening more feasible and cost-effective.Congenital cytomegalovirus (CMV) infection occurs in 0.2 to 1% of all births. In addition to infants with symptomatic cases, a proportion of asymptomatically infected newborns face a significant risk of late-onset sequelae (8,9,12,16,17,22,26). Since the early identification of congenitally infected newborns may lead to early intervention and antiviral treatment options (14), a simple and inexpensive assay for CMV detection is required to implement screening programs for congenital CMV infection (19).In spite of the progress in PCR-based assays, the need for DNA purification from body fluid specimens as well as the labor, costs, and other problems associated with specimen collection, transportation, and storage have limited the convenience of these assays. Although a small volume of urine can be used directly for PCR, inhibitors in urine reduce PCR efficiency (6,7,13,23). Robotic systems that may simplify the DNA purification process (20) are not affordable for every facility. The use of filter papers can resolve the problems associated with collection and storage, as exemplified by the use of dried blood spots (10). Washing filter papers has reduced the amount of inhibitors, which in turn has allowed for conventional PCR on the filter disk (25). Alternatively, DNA samples have been extracted and/or purified from specimens on filters and used for PCR (3,5,18,27). Although dried blood spots can be used for CMV screening (2), the assay may not detect some cases of congenital infection, as blood specimens contain smaller amounts of CMV than urine specimens (4, 11; N. Inoue and S. Koyano, unpublished results).In this study, we developed a real-time PCR assay using urine specimens on filter disks as a template for the reaction and demonstrated the assay's technological potential for screening for congenital CMV infection.First, we selected a filter paper on which PCRs could proceed efficiently (Fig. 1). Commercially available filter papers were spiked with dilutions of purified CMV, and filter disks obtained from the filters were added directly to PCR mixtures. Since Isocode filters allowed more efficient amplification than the others, these filters were used for the following experiments. Next, we found that only instruments equipped with a photomultiplier-tube scanning system (e.g., Stratagene MX3500P) could be used for real-time PCR assays with filter disks in the reaction mixture, due to the fact that instruments using a charge-coupled device camera (e.g., ABI7700) were adversely affected by nonspecific signals from the disks. The optimized real-time PCR conditions were as follows. Fifty microliters of reaction mixture contained 1ϫ Brilliant quantitative P...

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Storage of viruses on filter paper for genetic analysis

Cited by 22 publications

References 21 publications

Combination of Reverse Transcriptase PCR Analysis and Immunoglobulin M Detection on Filter Paper Blood Samples Allows Diagnostic and Epidemiological Studies of Measles

Combination of Reverse Transcriptase PCR Analysis and Immunoglobulin M Detection on Filter Paper Blood Samples Allows Diagnostic and Epidemiological Studies of Measles

Use of the Minimum Spanning Tree Model for Molecular Epidemiological Investigation of a Nosocomial Outbreak of Hepatitis C Virus Infection

Real-Time PCR Assay Using Specimens on Filter Disks as a Template for Detection of Cytomegalovirus in Urine

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