Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

In 1998, when the PCR technique was already popular, a Japanese company called Eiken Chemical Co., Ltd. designed a method known as the loop-mediated isothermal amplification of DNA (LAMP). The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets. Furthermore, the Bst DNA polymerase most used in LAMP shows a high strand displacement activity, which eliminates the DNA denaturation stage. One of the most significant advantages of LAMP is that it can be conducted at a stable temperature, for instance, in a dry block heater or an incubator. The products of LAMP can be detected much faster than in standard techniques, sometimes only requiring analysis with the naked eye. The following overview highlights the usefulness of LAMP and its effectiveness in various fields; it also considers the superiority of LAMP over PCR and presents RT-LAMP as a rapid diagnostic tool for SARS-CoV-2.

Freshwater mussels (Bivalvia: Unionidae) from the rising sun (Far East Asia): phylogeny, systematics, and distribution.

Freshwater mussels (Bivalvia: Unionidae) is a diverse family with around 700 species being widespread in the Northern Hemisphere and Africa. These animals fulfill key ecological functions and provide important services to humans. Unfortunately, populations have declined dramatically over the last century, rendering Unionidae one of the world's most imperiled taxonomic groups. In Far East Asia (comprising Japan, Korea, and Eastern Russia), conservation actions have been hindered by a lack of basic information on the number, identity, distribution and phylogenetic relationships of species. Available knowledge is restricted to studies on national and sub-national levels. The present study aims to resolve the diversity, biogeography and evolutionary relationships of the Far East Asian Unionidae in a globally comprehensive phylogenetic and systematic context. We reassessed the systematics of all Unionidae species in the region, including newly collected specimens from across Japan, South Korea, and Russia, based on molecular (including molecular species delineation and a COI + 28S phylogeny) and comparative morphological analyses. Biogeographical patterns were then assessed based on available species distribution data from the authors and previous reference works. We revealed that Unionidae species richness in Far East Asia is 30% higher than previously assumed, counting 43 species (41 native + 2 alien) within two Unionidae subfamilies, the Unioninae (32 + 1) and Gonideinae (9 + 1). Four of these species are new to science, i.e. Beringiana gosannensissp. nov., Beringiana fukuharaisp. nov., Buldowskia kamiyaisp. nov., and Koreosolenaia sitgyensisgen. & sp. nov. We also propose a replacement name for Nodularia sinulata, i.e. Nodularia breviconchanom. nov. and describe a new tribe (Middendorffinaiini tribe nov.) within the Unioninae subfamily. Biogeographical patterns indicate that this fauna is related to that from China south to Vietnam until the Mekong River basin. The Japanese islands of Honshu, Shikoku, Kyushu, Hokkaido, and the Korean Peninsula were identified as areas of particularly high conservation value, owing to high rates of endemism, diversity and habitat loss. The genetically unique species within the genera Amuranodonta, Obovalis, Koreosolenaiagen. nov., and Middendorffinaia are of high conservation concern.

Complete paternally inherited mitogenomes of two freshwater mussels Unio pictorum and Sinanodonta woodiana (Bivalvia: Unionidae).

Freshwater bivalves from the family Unionidae usually have two very divergent mitogenomes, inherited according to the doubly uniparental model. The early divergence of these two mitogenomic lineages gives a unique opportunity to use two mitogenomic data sets in a single phylogenetic context. However, the number of complete sequences of the maternally inherited mitogenomes of these animals available in GenBank greatly exceeds that of the paternally inherited mitogenomes. This is a problem for phylogenetic reconstruction because it limits the use of both mitogenomic data sets. Moreover, since long branch attraction phenomenon can bias reconstructions if only a few but highly divergent taxa are considered, the shortage of the faster evolving paternally inherited mitogenome sequences is a real problem. Here we provide, for the first time, complete sequences of the M mitogenomes sampled from Polish populations of two species: native Unio pictorum and invasive Sinanodonta woodiana. It increases the available set of mitogenomic pairs to 18 species per family, and allows unambiguous reconstruction of phylogenetic relationships among them. The reconstructions based on M and F mitogenomes which were separated for many millions of years, and subject to differing evolutionary dynamics, are fully congruent.

Hermaphroditic freshwater mussel Anodonta cygnea does not have supranumerary open reading frames in the mitogenome.

The complete mitogenome of Anodonta cygnea is 15,613 bp long. This compact, circular molecule contains the set of 37 genes, typical for invertebrate mitogenomes, in the same order and orientation as in maternally inherited genomes of other bivalves from the same subfamily. There are only two unassigned regions longer than 200 bp (266 bp and 274 bp) and no indication of any supranumerary open reading frames.

The complete maternal and paternal mitochondrial genomes of Unio crassus: Mitochondrial molecular clock and the overconfidence of molecular dating.

The availability of a rapidly growing number of complete mitochondrial genome sequences provokes high confidence dating approaches. However, even if the congruence between mitochondrial and nuclear markers is reasonable, the resulting topologies are frequently questionable. The unique opportunity to study the evolutionary history of two independent mitochondrial genomes in one phylogenetic context exists in the freshwater mussels family Unionidae. The two lineages function under doubly uniparental inheritance since well before the emergence of the family. Despite the relatively high number of available complete sequences of maternally inherited genomes, comparative analyses are limited by the small number of sequences of counterpart paternally inherited genomes. We have sequenced for the first time the representative set of five sequences (two maternal and three paternal) from the species Unio crassus. Comparative analysis of the phylogenies reconstructed using relevant mitogenomic data available in GenBank (13 species in total) reveal that single - genome inferences are congruent only if the relaxed clock is assumed.

Complete male mitochondrial genome of Anodonta anatina (Mollusca: Unionidae).

Anodonta anatina is a freshwater mussel of the family Unionidae. These mussels have a unique mitochondria inheritance system named doubly uniparental inheritance (DUI). Under DUI males have two, potentially very divergent mitochondrial genomes: F-type inherited from mother and M-type inherited from father. F-type is present in soma whereas M-type is present in gonadal tissues and sperm. Here we report two M-type sequences of complete mitochondrial genomes from Anodonta anatina. They are 16,906 bp long and their sequences are similar (0.1% divergence). The genome organization is identical to the other Unionidean M-type genomes published to date. There are 38 genes, including the recently described M-type specific M ORF. The presence of tRNA-like repeat in one of the noncoding regions, suggests that the control region is located in this area. Nucleotide composition is quite extreme, with AT content (66.2%) higher than in any other of the six published Unionidean M genomes.

Complete female mitochondrial genome of Anodonta anatina (Mollusca: Unionidae): confirmation of a novel protein-coding gene (F ORF).

Freshwater mussels are among animals having two different, gender-specific mitochondrial genomes. We sequenced complete female mitochondrial genomes from five individuals of Anodonta anatina, a bivalve species common in palearctic ecozone. The length of the genome was variable: 15,637-15,653 bp. This variation was almost entirely confined to the non-coding parts, which constituted approximately 5% of the genome. Nucleotide diversity was moderate, at 0.3%. Nucleotide composition was typically biased towards AT (66.0%). All genes normally seen in animal mtDNA were identified, as well as the ORF characteristic for unionid mitochondrial genomes, bringing the total number of genes present to 38. If this additional ORF does encode a protein, it must evolve under a very relaxed selection since all substitutions within this gene were non-synonymous. The gene order and structure of the genome were identical to those of all female mitochondrial genomes described in unionid bivalves except the Gonideini.

Distribution and genetic diversity of the terrestrial slugs Arion lusitanicus Mabille, 1868 and Arion rufus (Linnaeus, 1758) in Poland based on mitochondrial DNA.

The slugs Arion lusitanicus and Arion rufus inhabit ecologically degraded areas and are serious vegetation pests. In recent years, new localities of these species have been found in various parts of Poland. Here we study the morphology of 90 specimens from 9 populations of slugs. The morphology of the genital system allowed for the identification of 60 A. lusitanicus specimens from 6 populations and 30 A. rufus individuals from another 3 localities. In order to describe their genetic diversity at the level of the individual, population, and species, we compared sequences of the mitochondrial cytochrome oxidase subunit I (cox1) gene. The morphological analysis revealed that each of the studied populations comprised a single species, which was also confirmed by the molecular assay. We obtained 674-bp sequences of the cox1 gene for each species that showed a total of eight haplotypes. The genetic diversity of A. lusitanicus individuals ranged from 0.5% to 2.1%, whereas that of A. rufus was twice as low: 0.4-1.0%. The difference between the two species within the cox1 gene was at the level of 12%. Three A. lusitanicus and two A. rufus populations were found to be monomorphic. Large inter-population variability was found within each of the studied species, which suggests that the Polish populations of A. lusitanicus may have originated from repeated, separate introductions arriving from various parts of Europe.

Doubly uniparental inheritance of mitochondrial DNA in the freshwater bivalve Anodonta woodiana (Bivalvia: Unionidae).

Unlike the vast majority of organisms in which mitochondrial DNA is transmitted maternally (standard mitochondrial inheritance, SMI), some marine or freshwater bivalves exhibit a different pattern of mtDNA transmission, named doubly uniparental inheritance (DUI). In this case there are two types of mtDNA, i.e. the female-transmitted (F-type) and the male-transmitted (M-type), the latter being present only in the male gonads of Unionidae bivalves. Current knowledge on DUI does not cover any freshwater mussels that are found in Poland. This study confirms DUI ofmtDNA in A. woodiana, a Chinese mussel discovered in Poland in 1993. The sequence divergence in the COI gene region for the F-type ranged between 0% (separately for Polish and Japanese mussels) and 8.1% (between Polish and Japanese specimens). On the other hand, this parameter was higher for the M-type, reaching 9.7% between Polish and Japanese specimens. Sequence divergence between the F- and M-types reached 34-35% and, although very high, was still characteristic for the bivalves in which DUI had been found.

Genetic variability among freshwater mussel Anodonta woodiana (Lea, 1834) (Bivalvia: Unionidae) populations recently introduced in Poland.

The alien Chinese mussel Anodonta woodiana was first reported in Poland in the system of heated lakes near Konin in 1993. Genetic studies with use of three molecular techniques (isoenzyme electrophoresis, PCR-RFLP and sequence analysis of a COI gene fragment) were carried out on the Polish first populations of A. woodiana. The studies have revealed low genetic variation between the populations (Nei's genetic distance for 12 loci ranged 0.000 to 0.007) as well as their considerable polymorphism. Each population averaged 2.28 alleles per locus, 2.72 alleles per polymorphic locus, and 75% polymorphic loci. Restriction analysis of the COI gene fragment have not revealed variability between the analysed specimens, including males and females. Restriction enzymes, ScrFI, Csp6I, and EcoRI used in the COI gene fragment PCR-RFLP generate distinct restriction patterns, which can be molecular markers for A. woodiana. The sequence obtained for COI fragment was the same in the examined female and male specimens and represents F mitotype (DNA was isolated from somatic tissues). The divergence between A. woodiana F and M mitotypes is high (34%), however it remains within the range of the general character of the DUI (doubly uniparental inheritance) phenomenon in freshwater bivalves (Unionidae).

Mutations in the von hippel-lindau tumour suppressor gene in central nervous system hemangioblastomas.

Central nervous system hemangioblastomas (cHAB) are rare tumours which most commonly arise in the cerebellum. Most tumours are sporadic, but as many as one third of cHABs occur in the course of the hereditary disorder - von Hippel-Lindau disease (VHL). In order to diagnose new VHL families in Poland we performed sequencing of the entire VHL gene in archival material (paraffin embedded hemangioblastoma tissues) in a large series of 203 unselected patients with cHAB. VHL gene mutations were detected in 70 (41%) of 171 tumour samples from which DNA of relatively good quality was isolated. We were able to obtain blood samples from 19 of mutation positive cases. Eight (42%) of these harboured germline mutations in persons from distinct undiagnosed VHL families.