Specific Polymerase Chain Reaction Test Research Articles

Molecular and next-generation sequencing analysis of tick-borne pathogens of Rhipicephalus ticks (Acari: Ixodidae) in cattle and dogs

Ticks are small and adaptable arachnid ectoparasites and global carriers of various pathogens that threaten both human and animal health. They are present in many parts of China. A total of 858 ticks were collected from various regions and hosts, then subjected to species identification based on morphological and molecular characteristics, as described in the authors’ previous study. Eighty-three individual tick samples were selected for screening pathogens based on metagenomic next-generation sequencing (mNGS) and polymerase chain reaction (PCR) assays. The genomic DNA of tick species was extracted, and amplification of the bacterial 16S rRNA gene was carried out from DNA of individual ticks using V3-V4 hypervariable regions, before subjecting to metagenomic analysis. Each tick underwent specific PCR tests for identifying the bacterial species present, including Anaplasma, Ehrlichia, Coxiella, and Rickettsia, and also protozoans such as Babesia, Theileria, and Hepatozoon. Illumina NovaSeq sequencing results revealed that the dominant phylum and family in Rhipicephalus spp. were Bacteroidota and Muribaculaceae, respectively. Alpha diversity patterns varied depending on tick sex (R. linnaei only), species and location, but not on host. Furthermore, bacterial pathogens, including A. marginale (58 %, 29/50), A. platys (6 %, 3/50), E. minasensis (2 %, 1/50), Ehrlichia sp. (10 %, 5/50), T. sinensis (24 %, 12/50), T. orientalis (54 %, 27/50) and Coxiella-like bacteria (CLB) (80 %, 40/50) were detected in R. microplus, while E. canis (33.33 %, 10/30), H. canis (20 %, 6/30) and CLB (100 %, 30/30) were detected in R. linnaei. Also, Anaplasma sp. (33.33 %, 1/3), A. marginale (33.33 %, 1/3), R. felis (33.33 %, 1/3) and CLB (100 %, 3/3) were detected in R. haemaphysaloides. Dual and triple co-infections involving pathogens or CLB were detected in 84.00 % of R. microplus, 66.66 % of R. haemaphysaloides, and 33.00 % of R. linnaei. The report on microbial communities and pathogens, which found from Rhipicephalus spp. in Hainan Island, is an important step towards a better understanding of tick-borne disease transmission. This is the first report in the area on the presence of Anaplasma sp., A. marginale, R. felis and Coxiella, in R. haemaphysaloides.

Comparative studies on the detection of Theileria annulata infection by clinical, parasitological and molecular techniques in buffaloes (Bubalus bubalis)

During the period of 2019-2021, a total of 715 buffaloes were screened for Theileria infections. A set of clinical parameters were used to identify the theileriosis suspected animals which were later confirmed through microscopic tests (peripheral thin blood smear examination and lymph node biopsy smear examination) and molecular assay (cytochrome b based T. annulata specific polymerase chain reaction test). On the basis of clinical evaluation, 33.43% (239/715) buffaloes were suspected for the presence of theileriosis. Through laboratory examination, 63.60% (152/239), 7.48% (16/214) and 100% (239/239) were found positive for Theileria infection by blood smear examination, lymph node biopsy and PCR, respectively, among the clinically suspected theileriosis animals. The efficiency of PCR was recorded to be highest, followed by microscopic examination of peripheral blood smears, and the lowest value was obtained for lymph node biopsy smear examination. The results indicated that PCR is a highly sensitive diagnostic technique for confirmatory diagnosis of T. annulata parasitic infection in buffaloes, even at very low level of parasitemia. Analysis of the data based on thin blood smear examination revealed that the field incidence of theileriosis was 11.55% in the south-western region of Gujarat, India. The high incidences of T. annulata infection in buffaloes may be due to the sub-tropical climate of the region which is favorable for the growth and development of vector ticks and parasites.

Molecular test for detection of Mycoplasma ovipneumoniae associated with respiratory tract infection from goats in north and central parts of Kerala

Mycoplasmal pneumonia is an important contagious disease that significantly affects the economy of small ruminant farming worldwide and Mycoplasma ovipneumoniae (M. ovipneumoniae) is one of the major aetiological agents associated with pleuropneumonia in goats. It is considered as a serious epidemic disease of goats due to its huge economic impact and hence, rapid and early diagnosis of the disease is warranted. Clinical mycoplasmosis often lacks pathognomonic signs, so definitive diagnosis of the disease is quite burdensome. Polymerase chain reaction (PCR) test has been proven to be a specific and sensitive technique for the early diagnosis of mycoplasmosis. The present study highlights the detection of M. ovipneumoniae employing PCR test in 150 nasal swab samples collected from goats with symptoms of respiratory tract infection from five districts of Kerala. Results revealed that, out of 150 samples, 83 (55.33 per cent) were positive in 16S rRNA Mycoplasma genus specific PCR test. Among the 83 genus positive samples, 68 samples (45.33 per cent of total 150 samples) were positive in M. ovipneumoniae specific PCR test.

Human Papillomavirus-32-Associated Focal Epithelial Hyperplasia Accompanying HPV-16-Positive Papilloma-Like Lesions in Oral Mucosa

Human papillomavirus infection can cause a variety of benign or malignant oral lesions, and the various genotypes can cause distinct types of lesions. To our best knowledge, there has been no report of 2 different human papillomavirus-related oral lesions in different oral sites in the same patient before. This paper reported a patient with 2 different oral lesions which were clinically and histologically in accord with focal epithelial hyperplasia and oral papilloma, respectively. Using DNA extracted from these 2 different lesions, tissue blocks were tested for presence of human papillomavirus followed by specific polymerase chain reaction testing for 6, 11, 13, 16, 18, and 32 subtypes in order to confirm the clinical diagnosis. Finally, human papillomavirus-32-positive focal epithelial hyperplasia accompanying human papillomavirus-16-positive oral papilloma-like lesions were detected in different sites of the oral mucosa. Nucleotide sequence sequencing further confirmed the results. So in our clinical work, if the simultaneous occurrences of different human papillomavirus associated lesions are suspected, the multiple biopsies from different lesions and detection of human papillomavirus genotype are needed to confirm the diagnosis.

PM 7/42 (2) Clavibacter michiganensis subsp. michiganensis

Specific scopeThis standard describes a diagnostic protocol for Clavibacter michiganensis subsp. michiganensis.Specific approval and amendmentApproved in 2004‐09.Revision adopted in 2012‐09.

Focal epithelial hyperplasia (Heck's disease) in two Chinese females

Focal epithelial hyperplasia, or Heck's disease, is a relatively rare virus-induced benign disease. To the best of the authors’ knowledge it has not been reported in an ethnic Chinese population. The authors report two cases of focal epithelial hyperplasia (FEH) in Chinese patients, which were clinically and histologically in accord with FEH. In particular, the lesions in one case were located on the gingival mucosa, which is rarely affected by FEH. DNA extracted from paraffin-embedded specimens from the two patients was tested for the presence of human papilloma virus followed by specific polymerase chain reaction testing for 16, 18, 13, and 32 subtypes in order to confirm the clinical diagnosis.

A specific polymerase chain reaction test for the identification of Streptococcus pneumoniae

Using an approach based on the comparison of arbitrary primer polymerase chain reaction (PCR) genomic profiles from oral streptococci and Streptococcus pneumoniae strains, we identified a 434-bp genomic fragment apparently specific for S. pneumoniae. From the nucleotidic sequence of this common fragment, a pair of primers was designed and tested on a set of strains comprising the major Streptococcus species. One species, S. anginosus, gave an amplification product of the same length as S. pneumoniae. Sequence comparison of the S. anginosus and S. pneumoniae amplicons revealed several variations which were used to define a new set of primers giving a 181-bp S. pneumoniae–specific fragment. The amplified fragment contains the 5′ terminal part of a gene encoding a putative sugar-specific permease and an intergenic sequence. The PCR test was evaluated on 257 strains of invasive S. pneumoniae corresponding to clinical isolates and on 153 non-pneumoniae oral streptococci strains; in addition, 3 S. pseudopneumoniae strains were tested. With these primers, an amplification product was only obtained with the S. pneumoniae strains. Moreover, the test was successfully evaluated on 10 atypical S. pneumoniae strains related to pneumococcal diseases. In this study, we therefore established the capacity of a simple PCR test to discriminate S. pneumoniae from other Streptococci (including S. pseudopneumoniae), thus allowing rapid and accurate diagnosis.

Detection of human papilloma virus DNA in seven cases of focal epithelial hyperplasia in Iran

Focal epithelial hyperplasia (FEH), also known as Heck's disease, is a very rare disease of the oral cavity especially in Asia. It is a disease of children and young adults. Various causes have been implicated but in majority of cases FEH is caused by some subtypes of human papilloma virus (HPV) especially 13 and 32. Polymerase chain reaction (PCR) is a useful tool to identify HPV in FEH as it is a rapid and sensitive method. This study was designed to determine special HPV subtypes in seven cases of Heck's disease referring to our department by using PCR analysis. Paraffin sections of seven patients clinically diagnosed as FEH with compatible histhopathological features underwent DNA extraction procedures for PCR examination. Initially, all specimens were tested for presence of HPV virus followed by specific PCR testing for 16, 18, 13, and 32 subtypes in positive samples. Human papilloma virus was found in all samples. In five cases HPV13 and in one case HPV32 was positive. One case showed strong reactivity for HPV but none of tested subtypes were positive. All cases were negative for HPV 16 and 18. Similar with other studies about FEH, most of our cases were associated with HPV 13 but other subtypes may also be implicated.

Caracterización molecular de los genes histona H2A y ARNsno-Cl de Trypanosoma rangeli:: aplicación en pruebas diagnósticas

The application of polymerase chain reaction (PCR) to detect Trypanosoma cruzi and Trypanosoma rangeli often presents interpretation challenges. For example, some tests yield the amplification of similar bands from either parasite, polymorphic fragments of the same parasite, or present deviation towards T. cruzi in mixed infections.In this study, the basic researching needed for designing and standardizating specific PCR tests for each parasite species PCR are shown and analyzed. The TcH2AF/R primers were designed on the basis of the differential gene region observed between the histone h2a genic units of these parasites. These primers amplify a specific 234 bp fragment in T. cruzi (T. cruzi I and II strains). The TrF/R2 primers anneal to the intergenic regions of an 801 bp gene fragment encoding for six transcripts that conform the snoRNA-Cl cluster in T. rangeli. These primers amplify a fragment of 620 bp exclusively in KP1(-) and KP1(+) strains of the parasite.The application of these PCR tests in infected vectors and in chagasic patients show that both tests constitute useful tools for the diagnosis and differential identification of these trypanosomatids.

PCR detection of Toxoplasma gondii DNA versus serological diagnosis in women suffering from repeated abortion

Background: This study was conducted to test the utility of polymerase chain reaction (PCR) assay to detect recent infections with Toxoplasma in pregnant women. T.gondii DNA was detected by using B1 gene as a target for amplification which is highly specific for T.gondii and is well conserved among all of the tested strains. Results: This study revealed the following findings:(1) PCR was positive in 63 subjects, including 58 high risk cases (77.3%) and 5 of controls (12.5%). (2) 17 high risk cases (24.6%) had false positive IgM and 5 of controls (12.5%) had false negative result for IgM. (3) 17 high risk cases (32.7%) had false positive IgG and 5 of controls (12.5%) had false negative IgG. (4) No significant association between eating raw meat or contact with cats and positive ELISA for PCR but there is highly significant association between women with contact with soil and positive PCR. (5) No significant relation between residency and either ELISA or PCR. (6) Significant negative correlation between the age of the studied women and positivity of PCR. Conclusion: this study highlights the need for a confirmatory test to detect primary acute toxoplasmosis in pregnant women. It demonstrates the possibility of defining and selecting the high-risk cases for mother-to-child transmission of infection by combining specific serology and PCR tests to formulate a specific approach

PCR detection of Toxoplasma gondii DNA versus serological diagnosis in women suffering from repeated abortion

Background: This study was conducted to test the utility of polymerase chain reaction (PCR) assay to detect recent infections with Toxoplasma in pregnant women. T.gondii DNA was detected by using B1 gene as a target for amplification which is highly specific for T.gondii and is well conserved among all of the tested strains. Results: This study revealed the following findings:(1) PCR was positive in 63 subjects, including 58 high risk cases (77.3%) and 5 of controls (12.5%). (2) 17 high risk cases (24.6%) had false positive IgM and 5 of controls (12.5%) had false negative result for IgM. (3) 17 high risk cases (32.7%) had false positive IgG and 5 of controls (12.5%) had false negative IgG. (4) No significant association between eating raw meat or contact with cats and positive ELISA for PCR but there is highly significant association between women with contact with soil and positive PCR. (5) No significant relation between residency and either ELISA or PCR. (6) Significant negative correlation between the age of the studied women and positivity of PCR. Conclusion: this study highlights the need for a confirmatory test to detect primary acute toxoplasmosis in pregnant women. It demonstrates the possibility of defining and selecting the high-risk cases for mother-to-child transmission of infection by combining specific serology and PCR tests to formulate a specific approach

[Gene methylation patterns in digestive tumors].

The loss of tumor suppressor gene function damages the defensive mechanisms that protect the indemnity of genetic material. Promoter gene methylation is one of the inactivation mechanisms of suppressor genes. To study the methylation pattern of a group of genes in biopsy samples of gastrointestinal tumors. Forty eight gastric, 25 gallbladder, 24 colon and 6 pancreas cancer biopsy samples were randomly selected. The methylation pattern of CDH1, FHIT, CDKN2A, APC and MLH1 genes, was studied using a specific polymerase chain reaction test for methylation. Demographic, morphological and follow up variables of patients bearing the tumors were also analyzed. The general methylation frequency of CDH1, FHIT, CDKN2A, APC and MLH1 genes was 64.1, 56, 39.8, 18.1 and 34% respectively. In gastric cancer samples there was a correlation between APC gene methylation and well differentiated tumors; between CDH1 methylation and Lauren diffuse type and the presence of three or more metastasic lymph nodes; between FHIT, CDKN2A and CDH1 gene methylation and male gender. In less differentiated gallbladder tumors, the frequency of CDH1 methylation was higher. There was a tendency towards a lower survival in colon and gastric cancer when MLH1 (p =0.07) y CDKN2A (p= 0.06) were methylated, respectively. An abnormal methylation pattern was associated with morphological features in gastric and gallbladder cancer and with a tendency towards a lower survival in colon and gastric cancer.

Estudio de patrones de metilación génica en tumores del tubo digestivo

The loss of tumor suppressor gene function damages the defensive mechanisms that protect the indemnity of genetic material. Promoter gene methylation is one of the inactivation mechanisms of suppressor genes.To study the methylation pattern of a group of genes in biopsy samples of gastrointestinal tumors.Forty eight gastric, 25 gallbladder, 24 colon and 6 pancreas cancer biopsy samples were randomly selected. The methylation pattern of CDH1, FHIT, CDKN2A, APC and MLH1 genes, was studied using a specific polymerase chain reaction test for methylation. Demographic, morphological and follow up variables of patients bearing the tumors were also analyzed.The general methylation frequency of CDH1, FHIT, CDKN2A, APC and MLH1 genes was 64.1, 56, 39.8, 18.1 and 34% respectively. In gastric cancer samples there was a correlation between APC gene methylation and well differentiated tumors; between CDH1 methylation and Lauren diffuse type and the presence of three or more metastasic lymph nodes; between FHIT, CDKN2A and CDH1 gene methylation and male gender. In less differentiated gallbladder tumors, the frequency of CDH1 methylation was higher. There was a tendency towards a lower survival in colon and gastric cancer when MLH1 (p =0.07) y CDKN2A (p= 0.06) were methylated, respectively.An abnormal methylation pattern was associated with morphological features in gastric and gallbladder cancer and with a tendency towards a lower survival in colon and gastric cancer.

Molecular Diagnosis of Infective Endocarditis by Real-Time Broad-Range Polymerase Chain Reaction (PCR) and Sequencing Directly From Heart Valve Tissue

Traditionally, infective endocarditis (IE) has been microbiologically diagnosed by blood cultures or serology. However, conventional microbiologic methods do not always provide an etiologic diagnosis. We conducted the current study to evaluate the usefulness of a universal real-time polymerase chain reaction (PCR) of the 16S rRNA gene followed by sequencing for the diagnosis of IE in explanted heart valve tissue (HV) as part of the routine of a clinical microbiology laboratory, and to compare it with conventional culture of blood or HV. We prospectively analyzed 177 HV samples by universal PCR and sequencing: 48 were from 35 patients with definite IE and 129 were from 120 patients without IE. Specific PCR tests were used when necessary to confirm broad-range PCR results. For the 35 patients with IE, all of the HV samples except for 2 from the same patient gave positive PCR results. The microorganisms identified matched those isolated by blood culture in 31 cases. The other 3 patients had negative blood culture IE, but PCR made possible the detection of Tropheryma whipplei, Bartonella quintana, and Streptococcus gallolyticus. For the negative control group, universal PCR was completely negative in 123 of the 129 samples. Sensitivity, specificity, and negative and positive predictive values of this real-time PCR method were 96%, 95.3%, 98.4%, and 88.5%, respectively, for the diagnosis of IE, using the Duke criteria to define IE and using blood culture results to identify etiologic microorganisms. Conventional HV culture correlated poorly with blood cultures and molecular techniques, and frequently represented tissue contamination resulting from valve handling. Our universal PCR method has proved to be more sensitive, specific, and rapid than conventional culture methods, and should therefore be included as a new major Duke criterion for the diagnosis of IE. According to the results of the current study, this technique should be used to supplement blood and HV culture. Conventional HV cultures are frequently responsible for false-positive and false-negative results, and are not always useful to establish the etiology of IE.

Use of a polymerase chain reaction to subtype Mycobacterium avium subspecies paratuberculosis, an increasingly important pathogen from farmed deer in New Zealand

AIMS: To review the number of microbiologically-confirmed cases of Johne's disease in farmed deer since 2000, and determine the prevalence of the bovine and ovine subtypes of Mycobacterium avium subsp paratuberculosis (M. paratuberculosis), using a highly specific polymerase chain reaction (PCR) test on samples from infected herds. METHODS: The number of cases of M. paratuberculosis in farmed deer identified by culture or IS900 PCR was documented. A highly specific PCR test was applied to subtype M. paratuberculosis from BACTEC 12B cultures selected on the basis of one culture per deer herd, to give a wide coverage of herds in New Zealand. RESULTS: From January 2001 to October 2005, M. paratuberculosis was isolated from 1,141 farmed deer, and has now been identified by microbiological testing in over 600 deer herds in New Zealand. The bovine subtype of M. paratuberculosis was shown by a highly specific PCR test to be present in 91/95 herds examined; the ovine subtype was found in the remaining four herds. CONCLUSIONS: Since 2000, there has been a substantial increase in both the number of microbiologically-confirmed cases of Johne's disease in farmed deer and the number of infected herds. Johne's disease is now widespread and common in deer herds throughout New Zealand. Whilst the bovine subtype of M. paratuberculosis predominates in deer herds in New Zealand in which Johne's disease has been confirmed, the occasional finding of the ovine subtype highlights the need to consider both sheep and cattle as potential sources of infection for farmed deer.

Diagnostic Value of Bronchoalveolar Lavage in Community-acquired Pneumonia in a Routine Setting: A Study on Patients Treated in a Finnish University Hospital

Only a few previous studies have focused on the use or bronchoalveolar lavage (BAL) in patients with community-acquired pneumonia (CAP). Our aim was to evaluate the diagnostic value of BAL in CAP in a routine clinical setting. 71 disease episodes were retrospectively analysed. The patients had undergone BAL for serious or slowly responding pneumonia. All procedures were performed during antimicrobial treatment of the patient. BAL fluid was cultivated for bacteria, fungi, and viruses. In 68 episodes, 1 or several specific polymerase chain reaction tests were performed. Only 1 (1.3%) quantitative bacterial culture was considered diagnostic for CAP, and indicated a change of antimicrobial treatment. The diagnostic yield increased to 9.8% when other methods were used. A respiratory virus was the only aetiology in 3 (6.0%) patients. In slowly responding pneumonia, also hospital-acquired pathogens and malignancies were identified, resulting in a total diagnostic yield of 20.0%. Thus, even when a large array of diagnostic assays was applied, the value of BAL in pretreated patients with CAP was very small, and its therapeutic implications minimal. In a subgroup of slowly responding pneumonia, the procedure was of some usefulness even after commencement of antimicrobial treatment.

NEISSERIA MENINGITIDIS ENDOPHTHALMITIS: USE OF POLYMERASE CHAIN REACTION TO SUPPORT AN ETIOLOGIC DIAGNOSIS

We report a case of endogenous endophthalmitis in which the suspected etiology, Neisseria meningitidis, was supported by use of a specific polymerase chain reaction test on vitreous humor. This technology may be especially valuable in the future for those cases in which systemic and eye cultures are nonrevealing.

A rapid screening method for hepatocyte nuclear factor 1 alpha frameshift mutations; prevalence in maturity-onset diabetes of the young and late-onset non-insulin dependent diabetes.

Non-insulin dependent diabetes (NIDDM) is a polygenic heterogeneous disorder of glucose homeostasis. Maturity-onset diabetes of the young (MODY) is a monogenic subtype of NIDDM characterised by early-onset (< 25 years) and autosomal dominant inheritance. Mutations in the hepatocyte nuclear factor 1 alpha (HNF-1 alpha) gene have recently been shown to cause MODY. The incidence of mutations in this gene in MODY and late-onset NIDDM is not known. We have developed a rapid specific polymerase chain reaction test for HNF-1 alpha mutations; this test involves the use of fluorescently labelled forward primers and modified reverse primers to detect length polymorphisms resulting from frameshift mutations. With this method, we have screened 102 MODY probands, viz. 60 defined according to strict diagnostic criteria (autosomal dominant inheritance and at least one member diagnosed age < 25 years) and 95 late-onset NIDDM probands (diagnosed 35-70 years with > or = 1 affected relative), for the presence of 9 known HNF-1 alpha frameshift mutations, including 6 that occur at two sites for recurring mutation (residues 291/292 and 379). Mutations were detected in 11 of the strictly defined MODY probands and one mutation was also found in a single subject with early-onset NIDDM but no family history of the disease. The HNF-1 alpha frameshift mutations were not detected in any late-onset NIDDM subjects, suggesting these mutations do not have a major role in the pathogenesis of NIDDM. Our results indicate that the prevalence of the nine frameshift mutations in strictly defined UK MODY is 18%, with the P291fsinsC mutation alone having a frequency of 13%.

Taxonomy of Kudoa species (Myxosporea), using a small-subunit ribosomal DNA sequence

Myxosporeans in the genus Kudoa infect the flesh of many marine fishes and often cause unsightly lesions and softening of the flesh texture. We are particularly interested in K. thyrsites because it is associated with soft flesh in the Atlantic salmon (Salmo solar), an important commercial species in Canada. Sequences of the small-subunit (SSU) rDNA (about 1600 base pairs) were obtained from K. miniauriculata, K. amanuensis, and K. poniformis. We aligned these sequences with one obtained from coho salmon (Oncorhynchus kisutch) and designed "Kudoa general" primers (KUD1f and KUD2r). These primers, in combination with other general primers, were then used to obtain the SSU rDNA sequence of K. thyrsites from two host species, Atlantic salmon and tubesnout (Aulorhynchus flavidus), from British Columbia, Canada. Sequence comparisons of these isolates indicated that Kudoa species cluster by geographic location rather than by morphology of spores. The three species from the eastern Pacific were approximately 97% identical, whereas K. amamiensis (from Japan) was about 91% identical with these species. Sequence comparisons of K. thyrsites from Atlantic salmon and tubesnout revealed a difference of only 0.07% between these isolates. Comparison of SSU rDNA sequences from the four Kudoa species and Henneguya salminicolo analyzed in this study with those from other available myxosporean genera (Myxidium and Myxoholus) showed that taxonomic divisions at the order and suborder levels were consistent with classical views of the taxonomy of the Myxosporea. Using specific regions of the SSU rDNA, we also developed a sensitive and specific polymerase chain reaction test for detection of K. thyrsites.

Specific diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in dried blood spots by a polymerase chain reaction (PCR) assay detecting a point-mutation (G985) in the MCAD gene

The discovery of a point-mutation, adenine-to-guanine, at position 985 in the gene coding for MCAD (G985), gave the basis for an easy and specific polymerase chain reaction test. We tested the specificity of such a PCR based assay and detected correctly G985 and A985 in sequence verified cDNA clones. We showed that the G985 mutation is present in genomic DNA from 48 of 50 patients with confirmed MCAD deficiency, originating from various European countries, Australia and the USA. On the basis of this high frequency of the G985 mutation among patients, we improved and optimized the assay with respect to reliability and convenience for routine diagnostic and screening purposes. As little as 2 μl blood from filter-paper blood-spots (Guthrie spots) is sufficient for the test.