Nested Primers Research Articles

Uracil base PCR implemented for reliable DNA walking

PCR-based DNA walking is of efficacy for capturing unknown flanking genomic sequences. Here, an uracil base PCR (UB-PCR) with satisfying specificity has been devised for DNA walking. Primary UB-PCR replaces thymine base with uracil base, resulting in a primary PCR product composed of U-DNAs. A single-primer (primary nested sequence-specific primer) single-cycle amplification, using the four normal bases (adenine, thymine, cytosine, and guanine) as substrate, is then performed on the primary PCR product. Clearly, only those U-DNAs, ended by the primary nested sequence-specific primer at least at one side, will produce the corresponding normal single strands. Next, the single-cycle product undergoes uracil-DNA glycosylase treatment to destroy the U-DNAs, while the normal single strands are unaffected. Afterward, secondary even tertiary PCR is performed to exclusively enrich the target product. The feasibility of UB-PCR has been checked by obtaining unknown sequences bordering the three selected genetic sites.

Rapid detection, quantification and speciation of Leishmania using real-time PCR and DNA sequencing at the rRNA Internal Transcribed Spacer 2.

The ability to discriminate infection between closely related Leishmania species within the Viannia species complex, specifically L. braziliensis, L. guyanensis and L. panamensis is critical to inform the clinical diagnosis and determine the most efficacious treatment modality. We designed a nested primer set targeting the rRNA Internal Transcribed Spacer 2 (ITS2), located on Chromosome 27, to distinguish among all human infective Leishmania species. Separate nested and single primer pairs were developed for conventional and quantitative PCR approaches respectively. Species-specific single nucleotide polymorphisms and indels located within the PCR products were identified by Sanger sequencing. This single locus approach provides a sensitive and specific platform to identify the species of Leishmania causing infection.

Establishment of Nested PCR for the Detection of Pseudomonas plecoglossicida and Epidemiological Survey of Larimichthys crocea in the Southeast Coastal Region.

The visceral white nodules disease in the internal organs of Larimichthys crocea has caused significant harm in the aquaculture of this species, with Pseudomonas plecoglossicida considered one of the core pathogens causing this disease. In this study, we designed three pairs of specific nested PCR primers targeting the sctU gene of P. plecoglossicida, a crucial component of the Type III secretion system (T3SS), which is instrumental in bacterial pathogenesis and virulence. Through the optimization of PCR reaction conditions, specificity testing, and sensitivity determination, a method was established for the accurate detection of P. plecoglossicida. This method yielded single amplification products, exhibited a false positive rate of zero for reference bacteria, and achieved a detection sensitivity of a minimum of 2.62 copies/reaction for the target sequence. Using the detection method, we conducted analyses on the diseased populations of L. crocea, involving a total of 64 screened fishes along the southeast coast of China from 2021 to 2023. The results revealed that the infection rate of P. plecoglossicida in diseased L. crocea exceeded over 90% in March and April, while in other months, the maximum recorded infection rate was merely 10%. The detection method developed in this study shows potential for early warning and routine monitoring of visceral white nodules disease in the internal organs of species such as L. crocea.

Detection of circovirus in free-ranging brown rats (Rattus norvegicus)

Accidentally found, two poisoned brown rats from Hungary were surveyed for presence of circoviral DNA, using specific nested primers, designed against the rep gene of the virus. Both specimens were positive. The whole genomes were amplified using inverse PCR based on the Rep sequence parts and sequenced by the primer walking method. Genomic analyses revealed that these novel rat viruses, together with tawny owl-associated circovirus reported by Italian researchers in 2022, are sequence variations of the same virus from genus Circovirus. In phylogenetic reconstructions, these circovirus strains detected from brown rats clustered closest to circoviruses derived from faeces samples of various predatory mammals. Molecular data as well as the phylogenetic analyses of the complete derived replication-associated protein and the capsid protein, as well as the prey preference of the host species of the recently described tawny owl-associated virus suggest that brown rat could be the evolutionary adapted host of the viruses described in this paper (brown rat circovirus types 1 and 2) and the previously reported tawny owl-associated virus. Possible pathogenic and zoonotic role of these viruses need further studies.

Molecular diagnosis of echinococcosis in patients based on frozen paraffin tissue samples and fixed formalin and hydatid cysts isolated from livestock in a slaughterhouse.

Various genotypes of Echinococcus granulosus have been studied in high-disease-risk areas and identified as causative agents of cystic echinococcosis (CE). This study was performed to examine and identify the molecular hydatid cyst in the dissected human specimens in paraffin tissue, and the dissected animal cyst was characterized using the DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer 1 (ITS1). To determine the molecular properties of E. granulosus, 20 hydatid cyst samples (including 6 sheep samples, 9 camel samples, and 10 human paraffin samples) were collected from Zahedan and Zabol cities. After DNA extraction, molecular PCR was performed, and RFLP was evaluated. In this study, the Taq1 endonuclease cleavage enzyme was used. The patterns of DNA bands found in the isolates from human CE and animal bladder cysts were the same, as indicated by the results of ribosomal DNA-ITS1 amplification from E. granulosus. Two nested primer pairs were used. The rough size of the enhanced ITS1 piece was 444 and 391 base pairs (bp), individually. After cutting the PCR product with the Taq1 enzyme, the patterns of the fragments revealed that the samples had two identical RFLP patterns. The aftereffects of this study showed that the parasite genotypes confined to sheep, camels, and people had hereditary changes. The transcendent type of E. granulosus sensu lato in the area is E. granulosus sensu stricto, which featured the meaning of the sheep/canine cycle in human transmission. Albeit the band profile in the camel is now and again like the sheep strain, RLFP can be recognized utilizing the PCR strategy, and two differentiating band profiles using the chemical were found in this review.

Newly outbreak of Nipah virus: epidemiology, symptoms, transmission, diagnostic testing, treatment, and global health concern

Newly outbreak of Nipah virus: epidemiology, symptoms, transmission, diagnostic testing, treatment, and global health concern

Development of an ultra-sensitive single-tube nested PCR assay for rapid detection of Campylobacter jejuni in ground chicken

Traditional culture-based detection methods for Campylobacteri jejuni, a leading cause of human bacterial gastroenteritis worldwide, are time-consuming, cumbersome, and lacking in reliability. While polymerase chain reaction (PCR) has been frequently used for pathogen testing, it might generate false-negative results due to inadequate sensitivity. This study was the first to explore novel single-tube nested PCR (STN-PCR) to detect pathogens in food. Two pairs of nested PCR primers were designed based on the hippuricase gene of C. jejuni. The annealing temperatures and concentrations of nested primers were optimized to ensure the sequential use of outer and inner pairs of primers during amplification. Efficacy of the developed STN-PCR assay was compared with standard culture-based methods and conventional PCR in artificially contaminated ground chicken homogenate. Limit of detection of the STN-PCR assay was determined to be 3.6 × 101 CFU/ml of C. jejuni in the homogenate without enrichment, which was 100 times lower than conventional PCR. Moreover, 0.1 CFU/g of C. jejuni in ground chicken homogenate was identified by STN-real time PCR (rtPCR) after 24 h of enrichment, while a 48-h enrichment was required for culture-based methods and conventional rtPCR. This developed assay provides a powerful tool for rapid, highly specific, and ultra-sensitive detection of C. jejuni and may potentially be used to identify contaminated chicken and other foods.

Borrelia burgdorferi and Borrelia miyamotoi in Atlantic Canadian wildlife.

Borrelia burgdorferi and Borrelia miyamotoi are tick-vectored zoonotic pathogens maintained in wildlife species. Tick populations are establishing in new areas globally in response to climate change and other factors. New Brunswick is a Canadian maritime province at the advancing front of tick population establishment and has seen increasing numbers of ticks carrying B. burgdorferi, and more recently B. miyamotoi. Further, it is part of a region of Atlantic Canada with wildlife species composition differing from much of continental North America and little information exists as to the presence and frequency of infection of Borrelia spp. in wildlife in this region. We used a citizen science approach to collect a wide range of animals including migratory birds, medium-sized mammals, and small mammals. In total we tested 339 animals representing 20 species for the presence of B. burgdorferi and B. miyamotoi. We have developed new nested PCR primers and a protocol with excellent specificity for detecting both of these Borrelia species, both single and double infections, in tissues and organs of various wildlife species. The positive animals were primarily small non-migratory mammals, approximately twice as many were infected with B. burgdorferi than B. miyamotoi and one animal was found infected with both. In addition to established reservoir species, the jumping mouse (Napaeozapus insignis) was found frequently infected; this species had the highest infection prevalence for both B. burgdorferi and B. miyamotoi and has not previously been identified as an important carrier for either Borrelia species. Comprehensive testing of tissues found that all instances of B. burgdorferi infection were limited to one tissue within the host, whereas two of the five B. miyamotoi infections were diffuse and found in multiple systems. In the one coinfected specimen, two fetuses were also recovered and found infected with B. miyamotoi. This presumptive transplacental transmission suggests that vertical transmission in mammals is possible. This finding implies that B. miyamotoi could rapidly spread into wildlife populations, as well as having potential human health implications.

Ex vivo Antimicrobial Evaluation of Different Drugs Using RT-PCR Test

Introduction: Various medicaments are used in conjugation with mechanical cleaning and shaping to render root canals free of microbes. The different medicaments used tend to react among themselves sometimes resulting in deleterious products. For this reason use of alternative medicine, therapy is advocated.
 Aim: To evaluate the antimicrobial extent of different homeopathic drugs in comparison to the Ayurvedic and Allopathic medicines.
 Materials and Methods: A biofilm model of 8 mm dentine discs was prepared from single-rooted teeth, which were inoculated with E. faecalis for 21 days. 100 samples were divided into six groups Group I: Saline (negative Control), Group II: 2% Chlorhexidine Gluconate, Group III: Propolis, Group IV: RHUS Glabra, Group V: Zincum Oxydatum. The infected biofilm was then treated with the medicaments for seven days followed by testing to obtain threshold cycle (Ct) values using the Real-time polymerase chain reaction technique. DNA isolation was carried out and amplification was done using 16S rRNA gene sequence-based nested species-specific primers. DNA denaturation was done using DNA Thermocycler and a threshold cycle was obtained using Real-time Polymerize chain reaction technique. 
 Results: The threshold cycle (Ct) values obtained were subjected to statistical analysis that revealed a significant difference between the groups and among all 2% Chlorhexidine Gluconate, being the highest (30.3460±0.02505) followed by Propolis (30.1365±0.02621) then RHUS Glabra (30.0865±0.02581) better than Zincum Oxydatum (29.8070±0.02319) and least being Saline (29.6380±0.02505).
 Conclusion: All the homeopathic medicines showed antimicrobial properties comparable to the allopathic and ayurvedic drugs. Among RHUS Glabra and Zincum Oxydatum, RHUS Glabra performed significantly better.

Ladder-shape melting temperature isothermal amplification of nucleic acids.

A novel method, termed ladder-shape melting temperature isothermal amplification (LMTIA), was developed in this study. As a proof of concept, one pair of primers or two pairs of nested primers and a thermostable DNA polymerase were employed to amplify the internal transcribed spacerof Oryza sativa with the ladder-shape melting temperature curve. Our results demonstrated that the LMTIA assay with nested primers was 50-fold more sensitive than the LAMP assay with the same level of specificity. The LMTIA method has the potential to be used for the prevention and control of emerging epidemics caused by different types of pathogens.

Sex identification based on the CHD gene from Gentoo penguin (Pygoscelis papua) fecal DNA samples

We developed a reliable noninvasive PCR method for sex identification of the Gentoo penguin (Pygoscelis papua) based on the amplification of the chromobox-helicase DNA-binding (CHD) gene. Two sets of nested primers (F1/R1, F2/R2) were designed to amplify the introns of the CHD1W and CHD1Z genes. The fecal samples were lysed in an optimized lysis reagent containing PCR buffer. The fecal lysate was employed as the template DNA to perform PCR amplification. Following nested PCR, agarose gel electrophoresis was performed, and the amplicons exhibited a sex-specific band pattern (211 bp and 315 bp in females, 211 bp in males). There was no amplification failure of our specific primers in 14 tested samples (7 males and 7 females), and the results of genotypic identification matched the records of sex based on oviposition behavior and pairing combination (based on the monogamy of the Gentoo penguin). The preliminary results showed that these nested primers could also be used for sex identification of Chinstrap penguins (Pygoscelis antarctica) and King penguins (Aptenodytes patagonicus). In conclusion, we successfully sexed Gentoo penguins using DNA extracted from fecal samples and performed nested PCR of sex-specific CHD genes.

Toll-like Receptor 9 Agonists in HPV Vaccine Gardasil9

Gardasil9 is a recombinant human papillomavirus (HPV) 9-valent vaccine, containing purified major capsid L1 protein of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 re-assembled into virus-like particles (VLPs) as the active ingredients. Since the antigens are purified recombinant proteins, in theory Gardasil9 needs a potent adjuvant to generate high and sustained levels of antibodies. Historically, amorphous aluminum hydroxyphosphate sulfate (AAHS), listed as the adjuvant for Gardasil9, was known to require one or more Toll-like receptor agonists, such as the phospholipids in the recombinant hepatitis B vaccine, Recombivax HB®. However, there are no phospholipids in the purified HPV L1 proteins or in Gardasil9. But the Food and Drug Administration (FDA) reports that Gardasil4 does contain recombinant HPV L1-specific DNA fragments, and they may serve as Toll-like receptor 9 agonists in Gardasil9. The author has tested 5 samples of Gardasil9 from 4 manufacturing lots by PCR amplification with a set of degenerate primers followed by heminested PCR or by another 5 sets of non-degenerate nested PCR primers in an attempt to detect all 9 vaccine-relevant HPV type-specific L1 gene DNAs bound to AAHS in the vaccine. Sanger sequencing confirmed the presence of HPV 18, 11, 16 and 6 L1 gene DNA bound to insoluble AAHS nanoparticles, but they were unevenly distributed even within the same vaccine sample. Also, these fragments were at least partially in non-B conformations. Since no L1 gene DNA of HPV 31, 33, 45, 52, and 58 was amplified by the commonly used degenerate PCR primers, the results suggest that these may all be in non-B conformations or may have been removed as contaminants by a purification protocol. Further research is warranted to standardize the HPV DNA fragments in Gardasil which are known to be potent Toll-like receptor 9 agonists.

Toll-like Receptor 9 Agonists in HPV Vaccine Gardasil9

Gardasil9 is a recombinant human papillomavirus (HPV) 9-valent vaccine, containing purified major capsid L1 protein of HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58 re-assembled into virus-like particles (VLPs) as the active ingredients. Since the antigens are purified recombinant proteins, in theory Gardasil9 needs a potent adjuvant to generate high and sustained levels of antibodies. Historically, amorphous aluminum hydroxyphosphate sulfate (AAHS), listed as the adjuvant for Gardasil9, was known to require one or more Toll-like receptor agonists, such as the phospholipids in the recombinant hepatitis B vaccine, Recombivax HB®. However, there are no phospholipids in the purified HPV L1 proteins or in Gardasil9. But the Food and Drug Administration (FDA) reports that Gardasil4 does contain recombinant HPV L1-specific DNA fragments, and they may serve as Toll-like receptor 9 agonists in Gardasil9. The author has tested 5 samples of Gardasil9 from 4 manufacturing lots by PCR amplification with a set of degenerate primers followed by heminested PCR or by another 5 sets of non-degenerate nested PCR primers in an attempt to detect all 9 vaccine-relevant HPV type-specific L1 gene DNAs bound to AAHS in the vaccine. Sanger sequencing confirmed the presence of HPV 18, 11, 16 and 6 L1 gene DNA bound to insoluble AAHS nanoparticles, but they were unevenly distributed even within the same vaccine sample. Also, these fragments were at least partially in non-B conformations. Since no L1 gene DNA of HPV 31, 33, 45, 52, and 58 was amplified by the commonly used degenerate PCR primers, the results suggest that these may all be in non-B conformations or may have been removed as contaminants by a purification protocol. Further research is warranted to standardize the HPV DNA fragments in Gardasil which are known to be potent Toll-like receptor 9 agonists.

Specific detection and quantification of Ralstonia pseudosolanacearum race 4 strains from Zingiberaceae plant cultivation soil by MPN-PCR

The most probable number-polymerase chain reaction (MPN-PCR) method was assessed for the specific detection and quantification of Ralstonia pseudosolanacearum race 4 strains in soil from Zingiberaceae fields in Japan. Based on the genome sequence analysis of two bacterial strains [MAFF 211479 (type I) and MAFF 211472 (type II)] isolated from ginger, race 4 type I- and type II-specific nested PCR primer sets were designed, respectively. The MPN-PCR using these primer sets efficiently detected and quantified the pathogen in naturally and artificially infested soils.

Sequencing Herbarium Specimens of a Common Detrimental Plant Disease (Powdery Mildew)

Powdery mildew (Erysiphaceae) is a detrimental plant disease that occurs on a variety of economically important crops. Powdery mildew consists of over 873 species of fungal pathogens that affect over 10,000 plant species. Genetic identification of powdery mildew is accomplished using the internal transcribed spacer (ITS) and large subunit (LSU) regions of the nuclear ribosomal RNA gene cluster. The ITS and LSU regions of powdery mildews can be useful in ecological, epidemiological, phylogenetic, and taxonomic investigations. However, sequencing these regions is not without its challenges. For example, powdery mildew sequences are often contaminated with plant and/or fungal DNA. Also, there tends to be a limited amount and older specimens' DNA can fragment over time. The success of sequencing powdery mildew often depends on the primers used for running polymerase chain reaction (PCR). The primers need to be broad enough that they match the majority of powdery mildew DNA yet specific enough that they do not align with other organisms. A review of the taxonomy and phylogeny of the powdery mildews is presented with an emphasis on sequencing the ITS + LSU genomic regions. Additionally, we introduce a new nested primer protocol for sequencing powdery mildew herbarium samples that includes six new powdery mildew-specific primers. The new sequencing protocol presented allows specimens up to 130 years old to be sequenced consistently. Sequencing herbarium specimens can be extremely useful for addressing many ecological, epidemiological, phylogenetic, and taxonomic problems in multiple plant pathogenic systems including the powdery mildews.

Optimization of DNA extraction protocol using skeletal remains found in Sri Lanka

IntroductionThe preservation of DNA in old skeletal remains is reported to be very low in a tropical country like Sri Lanka due to prevailing climatic and environmental conditions such as high temperature, high rainfall and high humidity, etc. In this study, extraction of DNA from old skeletal remains dated back to 15 – 40 years was attempted by using previously published extraction protocols. Materials and MethodsA 15-years-old humerus (15Y) excavated from Kuliyapitiya area in Kurunegala district and the 40-yearsold tibia (40Y) received from Department of Anatomy, Faculty of Medical Sciences, University of Sri Jayewardenepura were used to extract old DNA. Human mitochondrial HVS I region of extracted DNA was amplified in PCR using four overlapping first round primers and second round nested primers respectively. A second-round nested PCR was performed. PCR amplification success was verified upon electrophoresis in 2% agarose gels. Results and AnalysisDNA bands were obtained with correct size ranges for all systems in both first and second round PCR products of amplified DNA extract of old bones 15Y and 40Y from modified phenolchloroform method. DNA bands were obtained from all four systems for 40Y bone DNA extract from DNA investigation Kit; QIAGEN, Germany. ConclusionIn the present study, we have successfully extracted and amplified DNA from old skeletal remains by using modified phenol chloroform method and DNA investigation Kit – QIAGEN, Germany, nevertheless the preservation of DNA in skeletal remains in Sri Lanka is very low.

TaqMan real-time and conventional nested PCR tests specific to yellow head virus genotype 7 (YHV7) identified in giant tiger shrimp in Australia

In 2013, a unique seventh yellow head virus genotype (YHV7) was detected in Black Tiger shrimp (Penaeus monodon) broodstock that suffered high mortality following their capture from Joseph Bonaparte Gulf (JBG) in northern Australia. To assist with its diagnosis and assessment of its distribution, prevalence and pathogenicity, YHV7-specific TaqMan real-time qPCR and conventional nested PCR primer sets were designed to ORF1b gene sequences divergent from the other YHV genotypes. Using high (≥108) copies of plasmid (p)DNA controls containing ORF1b gene inserts of representative strains of YHV genotypes 1–7, both PCR tests displayed specificity for YHV7. Amplifications of serial 10-fold dilutions of quantified YHV7 pDNA and synthetic ssRNA showed that both tests could reliably detect 10 genome copies. Pleopods/gills from wild P. monodon sourced from locations in geographically disparate regions across northern Australia as well as 96 juveniles (48 either appearing normal or displaying signs of morbidity) from a commercial pond experiencing mortalities were screened to partially validate the diagnostic capacity of the qPCR test. Based on these data and PCR primer/probe sequence mismatches with other newly identified YHV genotypes, both YHV7-specific PCR tests should prove useful in the sensitive detection and discrimination of this genotype from YHV 2 (gill-associated virus) and YHV6 that can occur in Australian P. monodon, as well as from YHV genotypes currently listed as exotic to Australia.

Screening nested-PCR primer for 'Candidatus Liberibacter asiaticus' associated with citrus Huanglongbing and application in Hunan, China.

Citrus Huanglongbing (HLB) is one of the most devastating citrus diseases worldwide. Sensitive and accurate assays are vital for efficient prevention of the spread of HLB-associated “Candidatus Liberibacter spp”. “Candidatus Liberibacter spp” that infect Citrus includes “Candidatus Liberibacter asiaticus” (Las), “Candidatus Liberibacter africanus” (Laf) and “Candidatus Liberibacter americanus” (Lam). Of them, Las is the most widespread species. In this study, a set of nested PCR primer pairs were screened to diagnose Las, and the nested PCR method greatly enhanced the sensitivity to detect Las up to 10 times and 100 times compared to qPCR and conventional PCR, respectively. Totally, 1112 samples from 5 different citrus cultivars in 39 different counties and cities were assayed by nested PCR. The results show that 384 samples were HLB-infected; the highest positive detection rate was 79.7% from the lopsided fruit samples, and the lowest positive detection rate was 16.3% from the apical dieback samples. The results indicate that the designed nested PCR primer pairs can detect Las from different symptomatic tissues, different citrus cultivars and different geographic regions. The set of nested PCR primers designed in the present study will provide a very useful supplementation to the current approaches for Las detection.

A pan-genotypic Hepatitis C Virus NS5A amplification method for reliable genotyping and resistance testing

BackgroundChronic infection with the Hepatitis C Virus (HCV) is associated with the risk of progressive liver disease. Although, HCV treatment options and viral cure rates have tremendously increased over the last decade, all currently licensed combination therapies contain inhibitors of the replication complex NS5A. Resistance-associated substitutions (RAS) in NS5A can limit the efficacy of therapy; however, resistance testing is routinely not recommended for all patients. Notably, pan-genotypic combinations have been approved, however the correct identification of the HCV genotype is still required for treatment decisions and is a good predictor for treatment success. ObjectiveThe aim of this study was the establishment of a pan-genotypic NS5A amplification method for reliable genotyping and simultaneous resistance testing in a fast and cheap routine diagnostic setup. Study designPan-genotypic degenerated nested PCR primer were designed and tested in 262 HCV-patients. The collection included samples from genotypes 1–7 and the median viral load was 1.07 × 106 IU/ml (range 248-21 × 106 IU/ml). ResultsAmplification of the expected 747bp fragment was successful in 257 of 262 (98.1%) samples including samples <1000 IU/ml. The direct comparison of the genotype information obtained with core sequencing to those obtained by NS5A prediction showed high concordance (97.3%) and discrepancies occurred only for relatively rare subtypes. Resistance analysis using Geno2Pheno[HCV] showed NS5A-RAS in 23 of 257 (8.9%) of samples. ConclusionsWe successfully developed a routine diagnostic method for pan-genotypic amplification of NS5A. This amplicon can be used for simultaneous genotyping and resistance testing for enhancing and improving routine HCV diagnostic.

Impact of placental malaria on adverse pregnancy outcomes in Sudanese women from Blue Nile state

Background: Sudan is one of the affected countries by malaria in sub-Saharan regions. Placental Malaria is a specific character of malaria infection during pregnancy. Sequestration of infected erythrocytes in the placental intervillous spaces is associated with adverse pregnancy outcomes, such as miscarriage, stillbirth, low birth weight (LBW) and congenital malaria. The aim of this study was to determine the impact of placental malaria on pregnancy outcomes in Blue Nile state, Sudan. Methods & Materials: A cross-sectional hospital based study was conducted during Jan. 2012- June 2014. Malaria infection was assessed at delivery time in peripheral maternal and neonatal blood, and in placental blood using microscopy and nested PCR. Finger prick blood was used for preparation of peripheral smears and dried spots. Smears were stained with Giemsa and examined microscopically for malaria parasites. DNA was extracted from the dried spots using chelex method and PCR was carried out using outer and nested primers. Results: A total of 336 mother–neonates pairs were enrolled in the study (mean maternal age 23.3 ± 6.3 yrs.). The mean (SD) haemoglobin was 10.1 (0.9) g/dl and the mean (SD) birth weight of the neonates was 2.4 (0.3) Kg. A high prevalence of P. falciparum infections was detected in maternal peripheral blood (30.5% and 42.7%) and placental blood (43.1% and 62.8%) using both microscopy and n-PCR respectively. The prevalence of congenital malaria was 2.0% by microscopy, while nested PCR identified positivity of 13.6%. Mothers of neonates with congenital malaria had at least one episode of clinical malaria in the index pregnancy. Placental malaria was significantly associated with low maternal haemoglobin level (P = 0.002), LBW (P = 0.011) and congenital malaria (P < 0.001). Conclusion: Infection with peripheral and/or placental malaria was associated with adverse pregnancy outcomes such as maternal anaemia and LBW. Materno- fetal transmission of P. falciparum is existence in Blue Nile state that may lead to intrautrine growth retardation, preterm birth and malaria illness mortality.