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Identification of the ‘Haryejosaeng’ mandarin cultivar by multiplex PCR-based SNP genotyping

Development of a PCR-RFLP method for detection of D614G mutation in SARS-CoV-2

In late 2019, an outbreak of respiratory disease named COVID-19 started in the world. To date, thousands of cases of infection are reported worldwide. Most researchers focused on epidemiology and clinical features of COVID-19, and a small part of studies was performed to evaluate the genetic characteristics of this virus. Regarding the high price and low availability of sequencing techniques in developing countries, here we describe a rapid and inexpensive method for the detection of D614G mutation in SARS-CoV-2. Using bioinformatics databases and software,

we designed the PCR-RFLP method for D614G mutation detection. We evaluated 144 SARS-CoV-2 positive samples isolated in six months in Northeastern Iran. Our results showed that the prevalent type is S-D in our isolates, and a small number of isolated belongs to the S-G type. Of 144 samples, 127 (88.2%) samples have belonged to type S-D, and 13 (9%) samples typed S-G. The first S-G type was detected on 2020 June 10. We have little information about the prevalence of D614G mutation, and it seems that the reason is the lack of cheap and fast methods. We hope that this method will provide more information on the prevalence and epidemiology of D614G mutations worldwide.

 

 

Identification of the ‘Haryejosaeng’ mandarin cultivar by multiplex PCR-based SNP genotyping

 

Most satsuma mandarin (Citrus unshiu Marc.) cultivars are difficult to identify in the seedling stage based only on morphological traits. Therefore, simple polymerase chain reaction (PCR)-based single-nucleotide polymorphism (SNP) markers were developed to specifically and rapidly distinguish the ‘Haryejosaeng’ cultivar, which is generally supplied to breeders of other satsuma mandarin cultivars. SNP markers were verified using high-resolution melt (HRM)-specific primers. PCR was performed to distinguish

 

‘Haryejosaeng’ from eight other satsuma mandarin cultivars using six SNP markers (P1-P6) specific for ‘Haryejosaeng’, with one negative control SNP primer pair. The best results were obtained using three SNP markers (P1, P2, and P5). In the multiplex PCR, markers P1, P2, and P5 yielded 165-, 150-, and 526-base pair amplicons, respectively, in ‘Haryejosaeng’, distinguishing it from other satsuma mandarin cultivars.

The selected SNP markers were validated by HRM with HRM-specific primers. The multiplex PCR with P1/P5 and P2/P5 also identified ‘Haryejosaeng’ obtained from a farm growing 17 different cultivars of satsuma mandarin. Specific SNP molecular markers were determined for accurately identifying the ‘Haryejosaeng’ cultivar by multiplex PCR to save the time and costs associated with its supply to breeders of satsuma mandarin.

Clinical Features and Temporal Changes of RT-PCR and Chest CT in COVID-19 Pediatric Patients

Objective: This work aims to investigate the clinical features and the temporal changes of RT-PCR and CT in COVID-19 pediatric patients. Methods: The clinical, RT-PCR, and CT features of 114 COVID-19 pediatric in-patients were retrospectively reviewed from January 21 to March 14, 2020. All patients had chest CT on admission and were identified as positive by pharyngeal swab nucleic acid test. The clinical features were analyzed, as well as the features and the temporal changes of RT-PCR and CT. Results: Fever (62, 54%) and cough (61, 54%) were the most common symptoms.

 

There were 34 (30%) cases of concurrent infections. The most common imaging features on CT were ground-glass opacities (46, 40%) and consolidation (46, 40%). The bilateral lower lobes were the most common pattern of involvement, with 63 cases (55%) involving one to two lobes, and in 32 (28%) cases CT was normal. Throughout the whole duration of COVID-19 in children, the diagnostic positive rate of RT-PCR has been far higher than that of CT (all P < 0.05). For RT-PCR follow-up, reliable negative results were obtained only 7 days after the onset of symptoms. Though lung involvement on chest CT progressed rapidly in several cases, lung involvement in children with COVID-19 is mild, with a median value of 2 on CT score. Conclusions: RT-PCR is more reliable than CT in the initial diagnosis of pediatric patients with COVID-19. On follow-up, reliable negative RT-PCR results are available 7 days after the initial symptoms. The use of CT should be considered for follow-up purposes only if necessary.

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Quantification of Ammonia Oxidizing Bacterial Abundances in Environmental Samples by Quantitative-PCR

Quantitative-PCR (qPCR) enables the quantification of specific DNA targets, such as functional or phylogenetic marker genes associated with environmental samples. During each qPCR cycle, the number of copies of a gene (or region) of interest in DNA samples is determined in real time using a fluorescence-based label and compared to a standard serial dilution.
Here, we describe a qPCR method to quantify the ammonia oxidizing bacteria involved in the first step of nitrification, using the amoA gene as a proxy of their abundance. The preparation of the standards from environmental samples and qPCR is presented in detail for specifically quantifying microbial abundance in environmental samples such as soil.

Peptide Nucleic Acid (PNA) Clamps to Reduce Co-amplification of Plant DNA During PCR Amplification of 16S rRNA Genes from Endophytic Bacteria

High-throughput sequencing of universal bacterial 16S rRNA gene (16S rDNA) amplicons is a routine method for characterizing bacterial diversity in a range of environments. For eukaryotic host-associated communities, however, plastid and mitochondrial genes are often co-amplified with, and greatly outnumber, bacterial 16S rDNA.
This makes it difficult to obtain sufficient numbers of target 16S rDNA sequences to characterize the diversity of endophytic bacterial communities. This chapter describes a method that improves the amplification of bacterial 16S rDNA from plant tissues by using a peptide nucleic acid (PNA) PCR clamp. The PNA clamp selectively binds to a targeted region of the plant genome and inhibits its amplification during PCR. PNA clamps are especially useful for characterizing bacterial communities on plant tissues with lower levels of microbial colonization such as the root tips and leaves.

Retrospective Post-mortem SARS-CoV-2 RT-PCR of Autopsies with COVID-19-Suggestive Pathology Supports the Absence of Lethal Community Spread in Basel, Switzerland, before February 2020

Introduction: Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread around the world. While the first case was recorded in Hubei in December 2019, the extent of early community spread in Central Europe before this period is unknown. A high proportion of asymptomatic cases and undocumented infections, high transmissibility, and phylogenetic genomic diversity have engendered the controversial possibility of early international community spread of SARS-CoV-2 before its emergence in China.
Methods: To assess the early presence of lethal COVID-19 in Switzerland, we retrospectively performed an analysis of deaths at University Hospital Basel between October 2019 and February 2020 (n = 310), comparing the incidence of clinical causes of death with March 2020 (n = 72), the month during which the first lethal COVID-19 cases in Basel were reported. Trends of COVID-19-suggestive sequelae, such as bronchopneumonia with organization, acute respiratory distress syndrome (ARDS), or pulmonary embolisms (PE) were evaluated.
In cases where autopsy was performed (n = 71), analogous analyses were conducted on the cause of death and pulmonary histological findings. Eight cases with a COVID-19-suggestive clinical history and histopathology between October 2019 and February 2020, and 3 cases before October 2019, were selected for SARS-CoV-2 RT-PCR.

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HCV6218 8 X 1 mL
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HCV6219 14 X 1 mL
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HCV6220 6 X 1 mL
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HCV6224 6 X 1 mL
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HCV6225 19 X 1 mL
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HCV6229 8 X 1 mL
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HCV9041 8 X 1 mL
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HCV9042 6 X 1 mL
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HCV9048 8 X 1 mL
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HCV9049 5 X 1 mL
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HCV Pre/Post Seroconversion Panel Donor# 66822 (5 X 1 mL)

HCV10235 5 X 1 mL
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HIV Seroconversion Panel Donor# 65404 (7 X mL)

HIV9013 7 X mL
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