Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of BK Virus

Department of Pediatrics, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA.
Journal of Clinical Microbiology (Impact Factor: 3.99). 06/2007; 45(5):1581-7. DOI: 10.1128/JCM.01024-06
Source: PubMed


Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity
and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity
in renal transplant recipients. The characteristics of the assay, including its specificity and sensitivity, were evaluated.
BKV LAMP was performed using various incubation times with a variety of specimens, including unprocessed urine and plasma
samples. A ladder pattern on gel electrophoresis, typical of successful LAMP reactions, was observed specifically only for
BKV and not for other viruses. The sensitivity of the assay with 1 h of incubation was 100 copies/tube of a cloned BKV fragment.
Additionally, a positive reaction was visually ascertained by a simple color reaction using SYBR green dye. BKV LAMP was also
successful for urine and plasma specimens without the need for DNA extraction. Due to its simplicity and specificity, the
LAMP assay can potentially be developed for “point of care” screening of BKV.

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    • "Alternative nucleic acid amplification (NAA) technologies, reviewed by Craw and colleagues [5] [6] [7], utilising isothermal conditions offer a range of potential advantages over PCR, including speed and simplicity, and lend themselves to near patient and point of care diagnostic testing [8] [9]. Loop-mediated isothermal amplification (LAMP) [10] is an example of an isothermal NAA technology that is typically faster than PCR [11] [12] [13] and reportedly less susceptible to common biological inhibitors [14] [15] [16] [17]. LAMP has been successfully utilised in the development of a wide range of diagnostic assays [18] [19] [20]. "
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    ABSTRACT: Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.
    Full-text · Article · Dec 2014
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    • "This virus can be diagnosed by a BKV blood test or a urine test for decoy cells. Polymerase chain reaction (PCR) techniques are often carried out to identify the virus [4]. The cornerstone of therapy is the reduction of immunosuppression. "
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    ABSTRACT: The BK virus is a member of the polyomavirus family. When the immune system is compromised, as in patients undergoing chemotherapy after hematopoietic stem cell and solid organ transplantation, the virus is reactivated, leading to haemorrhagic cystitis. While the BK virus is one of the most common causes of morbidity and mortality in patients after allogeneic stem cell transplantation, it rarely occurs after autologous stem cell transplantation. The early diagnosis and treatment of viral cystitis may prevent significant morbidity and mortality associated with haemorrhagic cystitis caused by the BK virus. It is not entirely clear how the BK virus affects prognosis in patients undergoing allogeneic and autologous hematopoietic stem cell transplantation.
    Full-text · Article · Sep 2014
    • "The pellets of the saliva samples were suspended in 200 ␮l PBS with 2% polyvinylpolypyrolidone and held overnight at −20 • C. Following Verweij's procedure (Verweij et al., 2001), the frozen sample was heated to 100 • C for 10 min before DNA extraction using the QIAamp DNA Mini Kit (QIAGEN, Germany). The urine sample pellet was suspended in 200 ␮l PBS, and DNA was extracted with the QIAmp viral RNA kit (QIAGEN, Germany) to provide a better yield of DNA than would be obtained with the Mini-kit (Bista et al., 2007). The procedure was performed according to manufacturer's instructions , with the final incubation time extended to 5 min to boost the yield of DNA (Beiromvand et al., 2011). "
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    ABSTRACT: This study investigated loop-mediated isothermal amplification (LAMP) detection of Plasmodium falciparum and Plasmodium vivax in urine and saliva of malaria patients. From May to November 2011, 108 febrile patients referred to health centers in Sistan and Baluchestan Province of south-eastern Iran participated in the study. Saliva, urine, and blood samples were analyzed with nested PCR and LAMP targeting the species-specific nucleotide sequence of small subunit ribosomal RNA gene (18S rRNA) of P. falciparum and P. vivax and evaluated for diagnostic accuracy by comparison to blood nested PCR assay. When nested PCR of blood is used as standard, microscopy and nested PCR of saliva and urine samples showed sensitivity of 97.2%, 89.4% and 71% and specificity of 100%, 97.3% and 100%, respectively. LAMP sensitivity of blood, saliva, and urine was 95.8%, 47% and 29%, respectively, whereas LAMP specificity of these samples was 100%. Microscopy and nested PCR of saliva and LAMP of blood were comparable to nested PCR of blood (к=0.95, 0.83, and 0.94, respectively), but agreement for nested PCR of urine was moderate (к=0.64) and poor to fair for saliva LAMP and urine LAMP (к=0.38 and 0.23 respectively). LAMP assay showed low sensitivity for detection of Plasmodium DNA in human saliva and urine compared to results with blood and to nested PCR of blood, saliva, and urine. However, considering the advantages of LAMP technology and of saliva and urine sampling, further research into the method is worthwhile. LAMP protocol and precise preparation protocols need to be defined and optimized for template DNA of saliva and urine.
    No preview · Article · Apr 2014 · Acta tropica
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