ABSTRACT
Objective:To investigate the accuracy, effectiveness and feasibility of MassARRAY genotyping assay in the diagnoses of neonatal genetic metabolic diseases.Methods:This is a retrospective study. From December 2016 to January 2020, newborns were screened by tandem mass spectrometry at the Zhejiang Newborn Screening Center, among which the data of 7 922 suspected positive cases of genetic metabolic diseases were collected. These patients were then tested for the common variants of 27 genetic metabolic diseases by MassARRAY genotyping assay, along with further testing using Sanger or next-generation sequencing used to verify and/or further search for potential variants.Results:A total of 1 408 cases were tested with MassARRAY. Among these, 307 cases were confirmed with certain genetic metabolic diseases. The detection rate of hyperphenylalaninemia was the highest, followed by primary carnitine deficiency, short acyl-coA dehydrogenase deficiency and methylmalonic acidemia. With these cases, the consistency of Sanger sequencing and MassARRAY was 100% (307/307). Another 287 cases were identified as carriers by MassARRAY with a 49.1% (141/287) consistency in reference to Sanger sequencing, mainly involving SLC22A5 and MCCC1 genes. Meanwhile, 50.8% (146/287) of these cases were found to have another variant mainly involving PAH, PTS and ACADS genes. The remaining 814 cases have no variants; 158 cases out of these patients have continuously abnormal amino acids, acyl carnitines, urine organic acid and/or other biochemical indices, and were tested by next-generation sequencing, among which 38% (60/158) were detected with two variants. In this study, a total of 513 patients with genetic metabolic disease were diagnosed, and the detection rate of MassARRAY was 59.8% (307/513). Conclusions:MassARRAY genotyping assay can be used as an early molecular screening method for neonatal genetic metabolic diseases. The detection rate is particularly high in diseases with a high concentration of hotspot variants, such as hyperphenylalaninemia and primary carnitine deficiency. The future application value of MassARRAY should be further improved by continuously optimizing its ability to identify new disease genes and potential variable sites.
ABSTRACT
Even after the availability of effective anti-leprosy drugs, leprosy treatment is facing the challenge of emergence of Mycobacterium leprae strains resistant to dapsone, rifampicin and ofloxacin. As the conventional mouse foot pad (MFP) assay is time consuming and requires sufficient bacterial load, it is important to adapt, develop and use molecular assay(s) that can detect M. leprae strains resistant to the drugs. Real-time PCR (rPCR) assay targeting RLEP sequences was used for detection of M.leprae DNA. Further a nested PCR reaction technique was optimized using sets of primers targeting folP, rpoB and gyrA gene target. Amplicons were sequenced to detect mutations. The optimal reactions were applied to slit skin smear samples from 20 leprosy patients and was found to be applicable for slit skin smear samples. The optimized assay could genotype 17 M. leprae strains for drug resistance, all (100%) of these strains were found to be sensitive for dapsone and rifampicin and 5.9% (1/17) resistant for fluoroquinolones (ofloxacin). This genotyping test could be used to detect leprosy drug resistance and may be useful for patient care. It will be also be important to validate the assay developed in this study with mouse foot technique and compare it with other molecular assays developed by investigators from different countries and then choose the best for patient care/surveillance purposes.
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Background: Many human papillomavirus (HPV) types are associated with cervical cancer (CC). Therefore, HPV genotyping has both clinical and epidemiological importance. HPV 16 and 18 are two principal high-risk types responsible for more than 70% of all CC cases. Although several commercial and non-commercial genotyping assays are available, there is a need for a cost-effective and sensitive genotyping method for low- and middle-income countries. Methods: The study was aimed at evaluation of loop-mediated isothermal amplification (LAMP) assay for HPV genotyping in cervical samples. A total of six primer sets for each HPV type were selected for the assay. The LAMP assay was standardised and validated with HPV control panel. Cervical biopsies were subjected to nested multiplex polymerase chain reaction (NM-PCR; as a part of routine diagnostic workup) and LAMP (HPV 16 and 18) simultaneously. Results: A total of 225 clinical samples were processed during the study period. The sensitivity of the assay was determined using the 10-fold dilutions of positive controls. Both the HPV 16-LAMP and HPV 18-LAMP assays were shown to detect as low as 10 viral copies per reaction, which is similar to that of NM-PCR. The LAMP assay had a good agreement (new cases; 92%, post-chemoradiotherapy [post-CRT]; 89.1%) with NM-PCR for the detection of both HPV 16 and 18. As compared to histology (new cases; 79.8%, post-CRT; 51.3%), LAMP had better agreement with NM-PCR for detection of HPV from post-CRT cases. Conclusions: We evaluated the LAMP assay for simultaneous detection and typing of HPV 16 and 18. The assay had good agreement with NM-PCR for detection of both HPV 16 and 18. The LAMP assay is a promising tool for HPV genotyping along with routine cervical cytology, especially in resource-constrained settings.
ABSTRACT
Objective To explore the distributions of genotypes of HPV infection in CIN and cervical carcinoma. Methods Cervical exfoliated cells were collected from 365 patients with abnormal cervical histology , and subjected to genotyping assay. Results The most prevalent HPV types were 16, 18,52, 58 and 33. The prevalence ratio of HPV 33,52,58 was signi cantly lower in squamous cell carcinoma. Multiple infections decreased from CIN II/III to cervical cancer. Conclusion Besides HPV 16/18, the 52/58/33 subtypes are also important in the development of cervical cancer.