A novel loop-mediated isothermal amplification-based genotyping method and its application for identifying proprotein convertase subtilisin/kexin type 9 variants in familial hypercholesterolemia.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in regulating low-density lipoprotein levels in plasma. While PCSK9 variants are causatively associated with familial hypercholesterolemia (FH), additional genotyping methods for FH targeting PCSK9 variants are required in a clinical setting. Loop-mediated isothermal amplification (LAMP) is a unique amplification method that amplifies a target gene under isothermal conditions (60-65 °C). However, a robust standardized method has not yet been established for LAMP-based genetic screening tests for genetic diseases, including FH. The present study aimed to develop a novel modification of the LAMP method designed to genotype single nucleotide variants (SNVs) and to apply it for the detection of PCSK9 variants. METHODS: Using short quenching probes (≤ 10 nucleotides) for the loop structures of LAMP amplicons, accurate detection of SNVs was verified separately for each allele, without any additional procedures, within 3 h. The diagnostic performance of this method in detecting PCSK9 variants was validated in FH patients. RESULTS: All PCSK9 variants tested via conventional sequencing in FH patients were successfully detected using this novel LAMP method. CONCLUSIONS: We developed a LAMP-based genotyping method to detect PCSK9 variants in FH. Compared to conventional sequencing, our genotyping method is relatively convenient and time-efficient and is suitable for the screening of FH in clinical settings. Future studies on various genes are also warranted.

Fully Automated Molecular Diagnostic System "Simprova" for Simultaneous Testing of Multiple Items.

Nucleic acid amplification-based diagnostics is known as one of the molecular diagnostic systems that allows higher sensitive detection of pathogens than test methods such as immunoassay. However, it has not been widely used because it is complicated to use and takes a long time to generate results. On the other hand, development of fully automated molecular diagnostic systems has been growing around the world as demand for such systems from physicians and laboratory technicians has increased. To meet this demand, we have developed the "Simprova" fully automated molecular diagnostic system, which takes advantage of LAMP (Loop-mediated Isothermal Amplification), a method Eiken Chemical Co., Ltd. invented. Simprova comprises a master unit that controls the entire system and a test unit that extracts and purifies nucleic acid from samples (pretreatment), and uses the LAMP method to detect and amplify nucleic acid. Users can obtain test results automatically by simply installing a pretreatment cartridge, a multi-well testing chip and the sample in the test unit. The multi-well testing chip has 25 reaction wells connected by channels and enables simultaneous testing of multiple targets with one sample. Turnaround time for one test is approximately 30 minutes. Since a conventional extraction and purification method using magnetic-bead separation is used for the pretreatment, nucleic acid can be extracted from serum, plasma, whole blood, urine, and sputum, for example. In addition, the system can perform random-access testing by connecting four test units to the master unit to realize near-the-patient testing. Simprova is therefore a robust and useful system for a wide variety of applications.

Human papillomavirus infection in male patients with STI-related symptoms in Hanoi, Vietnam.

This cross-sectional study investigated the prevalence, genotypes, and risk factors for human papillomavirus (HPV) infection in Hanoi, Vietnam. The study included 192 males (mean age, 32.9 years) with symptoms related to sexually transmitted infections (STI). Urinary, penile, and urethral samples were collected in April and May, 2014. HPV DNA was detected with PCR, performed with modified and/or original GP5(+)/GP6(+) primers. HPV genotypes were determined with a gene array assay. Neisseria gonorrhoeae (NG) and Chlamydia trachomatis (CT) DNA were detected with loop-mediated isothermal amplification. HPV DNA, NG, and CT were detected in 48 (25.0%), 23 (12.0%), and 41 (21.4%) patients, respectively. HPV DNA appeared in penile samples (21.0%, 39/186) more frequently than in urinary (3.1%, 6/191, P < 0.001) and urethral (9.4%, 18/192, P = 0.002) samples. Among patients with HPV, genotype prevalence was: HPV81 (22.9%), HPV52 (18.8%), HPV18 (16.7%), and HPV16 (6.3%). Multiple-type and high risk-type HPV infections were determined in 33.3% and 64.6%, respectively. Multivariate analysis showed a significant association of HPV infection in urethra with younger sexual debut age. HPV52 was the most prevalent high-risk HPV genotype, whereas HPV16 was less common in the male Vietnamese patients with STI-related symptoms. Younger sexual-debut age was a risk factor for HPV infection in urethra.

Infection with high-risk HPV types among female sex workers in northern Vietnam.

Vaccines against two high-risk human papillomavirus (HPV) types, HPV-16, and HPV-18, are in use currently, with high efficacy for preventing infections with these HPV types and consequent cervical cancers. However, circulating HPV types can vary with geography and ethnicity. The aim of this study was to investigate the prevalence of HPV types and the association between HPV types and abnormal cervical cytology among female sex workers in Northern Vietnam. Cervical swabs and plasma samples were collected from 281 female sex workers at two health centers in Hanoi and Hai Phong in 2009. The HPV L1 gene was amplified by PCR using original and modified GP5(+)/6(+) primers. Amplified PCR products were genotyped by the microarray system GeneSquare (KURABO) and/or clonal sequencing. Of the 281 women, 139 (49.5%) were positive for HPV DNA. Among the HPV-positive samples, 339 strains and 29 different types were identified. Multiple-type and high risk-type HPV infections were found in 85 (61.2%) and 124 (89.2%) women, respectively. The most common genotype was HPV-52, followed by HPV-16, HPV-18, and HPV-58. Abnormal cervical cytology was detected in 3.2% (9/281) of the women, and all of these samples were positive for HPV-DNA. Age ≤25 years and infection with human immunodeficiency virus were associated positively with HPV infection among the women while ever smoking was associated negatively. These results show that HPV-52 is most prevalent among female sex workers in Northern Vietnam, most of whom had normal cervical cytology. This information may be important for designing vaccination strategies in Vietnam.

Oral and cervical human papillomavirus infection among female sex workers in Japan.

It has been reported recently that oral human papillomavirus (HPV) infection is associated with oropharyngeal squamous cell carcinomas. The aim of this study was to determine the prevalence of HPV infection and HPV types in the oral cavity and cervix of female sex workers in Japan. Oral and cervical swabs were taken from 196 female sex workers who visited a clinic for regular medical checkups in 2007, and genomic DNA was extracted from those specimens. The HPV L1 gene was amplified by polymerase chain reaction (PCR) using original and modified GP5(+)/6(+) primers, and genotyping was performed using the Kurabo GeneSquare Microarray or by sequencing cloned PCR products. HPV DNA was detected in the oral cavity of 12 (6.1%) women, with HPV-56 being the most common type (7/12). Likewise, HPV DNA was detected in the cervix of 103 (52.6%) women, with HPV-52 (30/103, 29.1%), followed by HPV-16 (24.3%) and HPV-56 (18.4%), being the most common. Of the 12 women with oral HPV infection, only two were infected with the concordant HPV genotype in the cervix. These findings suggest that oral HPV infection occurs independently of cervical HPV infection in this population, and that oral HPV infection may play a role in HPV transmission in Japan.

Rapid detection of human immunodeficiency virus type 1 group M by a reverse transcription-loop-mediated isothermal amplification assay.

A rapid one-step reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the pol-integrase gene was developed to detect human immunodeficiency virus type 1 (HIV-1) group M. This HIV-1 RT-LAMP assay is simple and rapid, and amplification can be completed within 35min under isothermal conditions at 60 degrees C. The 100% detection limit of HIV-1 RT-LAMP was determined using a standard strain (WHO HIV-1 [97/656]) in octuplicate and found to be 120 copies/ml. The RT-LAMP assay was evaluated for use for clinical diagnosis using plasma samples collected from 57 HIV-1-infected and 40 uninfected individuals in Cameroon, where highly divergent HIV-1 strains are prevalent. Of the 57 samples from infected individuals, 56 harbored group-M HIV-1 strains, such as subtypes A, B, G, F2, and circulating recombinant forms (CRFs) _01, _02, _09, _11, _13; all were RT-LAMP positive. One sample harboring group-O HIV-1 and the 40 HIV-1-uninfected samples were RT-LAMP negative. These findings indicate that HIV-1 RT-LAMP can detect HIV-1 group-M RNA from plasma samples rapidly and with high sensitivity and specificity. These data also suggest that this RT-LAMP assay can be useful for confirming HIV diagnosis, particularly in resource-limited settings.

Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay.

The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3' noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63 degrees C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.

A simple method for the detection of measles virus genome by loop-mediated isothermal amplification (LAMP).

Approximately 20,000-30,000 measles patients were reported in a surveillance of infectious diseases because of low vaccine coverage of 80% in Japan. Among them, some were thought to be secondary vaccine failure (SVF) with generally mild or non-typical measles illness and sometimes became a source of further transmission. We have developed a new, sensitive, and rapid method to detect the measles virus genome by reverse transcription loop-mediated isothermal amplification (RT-LAMP). We examined 50 nasopharyngeal secretion (NPS) samples that were obtained during the 1999 outbreak and stored at -70 degrees C and fresh NPS, lymphocytes and sera from 11 patients in 2003. Total RNA was extracted from the samples and subjected to reverse transcription-polymerase chain reaction (RT-PCR) and RT-LAMP. We detected the genomic RNA corresponding to at least 0.01-0.04 TCID50, 30-100 copies in samples by RT-LAMP within 60 min after extraction of RNA, and all four genotypes isolated in Japan were equally amplified. Specific DNA amplification was monitored spectrophotometrically by real time turbidimeter and the quantity of RNA was calculated. Measles virus genome was detected in 44 of 50 stored NPS by RT-PCR and in 49 by RT-LAMP. The vaccine strain was discriminated from wild strains after sequencing the LAMP products. RT-LAMP is a useful rapid diagnostic method for the detection of measles virus without any special apparatus, showing higher sensitivity than RT-PCR, and expected to be applied for hospital-based infection control and for laboratory-based measles surveillance.